EP1673450B1 - Vitamin k epoxide recycling polypeptide vkorc1, a therapeutic target of coumarin and their derivatives - Google Patents

Vitamin k epoxide recycling polypeptide vkorc1, a therapeutic target of coumarin and their derivatives Download PDF

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EP1673450B1
EP1673450B1 EP04790318A EP04790318A EP1673450B1 EP 1673450 B1 EP1673450 B1 EP 1673450B1 EP 04790318 A EP04790318 A EP 04790318A EP 04790318 A EP04790318 A EP 04790318A EP 1673450 B1 EP1673450 B1 EP 1673450B1
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Prior art keywords
vkorc1
polypeptide
sequence
activity
coumarin
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German (de)
French (fr)
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EP1673450A1 (en
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Johannes Oldenburg
Clemens R. MÜLLER-REIBLE
Andreas Fregin
Tim-Matthias Strom
Simone Rost
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Baxter Healthcare SA
Baxter International Inc
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Baxter Healthcare SA
Baxter International Inc
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Priority to PL04790318T priority Critical patent/PL1673450T3/en
Priority to EP10002316A priority patent/EP2189523B1/en
Priority to DK10002316.7T priority patent/DK2189523T3/en
Priority to PL10002316T priority patent/PL2189523T3/en
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Definitions

  • the invention relates to methods for identifying coumarin derivatives, and also claims VKORC1 polypeptides and VKORC1 nucleic acids containing a sequence abnormality associated with a VKORC1 associated deficiency such as warfarin resistance, wherein the VKORC1 polypeptides and VKORC1 nucleic acids can be used for diagnosing these deficiencies. Moreover, the invention relates to methods for identifying coumarin derivatives usable In pest control of rodents.
  • coumarin are widely used for the prevention of thrombosis such as in patients Immobilized after surgery, patients having a chronic heart failure, patients having atherosclerotic vascular disease, patients having a malignancy, and patients that are pregnant.
  • coumarins are the most widely used oral anticoagulants for the treatment and prophylaxis of thrombosis [Suttie, 1987].
  • Coumarins are typically derivatives of 6-hydroxycoumarin, such as 3-(acetonylbenzyl)-4- hydroxycoumarin (COUMADIN ® ).
  • the coumarins target the blood coagulation cascade indirectly by inhibition of the vitamin K cycle.
  • Vitamin K is an essential cofactor for the post-translational activation by gamma-carboxylation of a group of regulatory proteins, the Gla-proteins. In several metabolic pathways, some key proteins require carboxylation for proper function.
  • the blood coagulation cascade is the best-studied example. Here, the procoagulant factors II, VII, IX and X, and the anticoagulant factors protein C, protein S, protein Z are dependent on gamma-carboxylation.
  • This post-translational modification enables the attachment of the modified proteins - in the presence of calcium - to phospholipid- bilayer membranes which is an essential step in the activation of blood coagulation [Sperling et al., 1978][Esmon et al., 1975].
  • vitamin K1 hydroquinone vitamin K1H2
  • VKOR vitamin K-2,3-epoxide reductase
  • VKCFD vitamin K dependent clotting factors
  • Clinical symptoms of the disease include episodes of perinatal intracerebral hemorrhage sometimes with fatal outcome. The bleeding tendency is usually completely reversed by oral administration of vitamin K. Additional symptoms in newborns can resemble warfarin embryopathy with nasal and distal phalangeal hypoplasia and premature calcification of epiphyses[Pauli et al., 1987]. The disease may result either from a defective resorption/transport of vitamin K to the liver [Prentice, 1985] or from mutations in one of the genes involved in gamma-carboxylation.
  • a coumarin therapy sometimes induces skin necrosis in patients and if applied during pregnancy may cause embryopathy creating a need for novel coumarin derivatives which do not cause these effects.
  • Phenobarbital induce lower plasma levels of coumarins due to an increased metabolization of coumarin which is believed to be caused by the mixed-function oxidases like the cytochrome P450 mixed-function oxidases.
  • Such interaction is of clinical relevance if the appropriate regimen of e.g. Phenobarbital and coumarin has been determined and later on only administration of Phenobarbital is discontinued leading to a rise of the plasma level of coumarin which causing excessive anticoagulation.
  • Other drugs like Amiodarone cause a delayed metabolization of coumarin leading again to excessive anticoagulation if co-administered with coumarins. Since the molecules affected by coumarins are not known in the art there is a need to develop novel coumarins and tools to identify the latter in order to solve these problems.
  • WO 00/03015 describes nucleic acid and amino acid sequences of human transport protein homologs and the use thereof (cf. for examples page 1, lines 5 to 7). Further, the Database EMBL, Mus musculus cDNA library (XP002318821) describes the cDNA of a Mus musculus 13 days embryo head (cf. for example the title). Additionally, Fregin, A. et al, Blood, Vol. 100, No. 9, 1 November 2002 , describes the assignment of a second gene for hereditary combined multiple coagulation factor deficiency to chromosome 16 (cf. for example page 3231, left hand column, lines 22 to 25) and Zimmermann, A. et al., Biochemical Pharmacology, Vol. 23, pp. 1033-1040 , describes the influence of warfarin on prothrombin formation and the regeneration of vitamin K from vitamin K-2,3-epoxide (cf. for example the abstract).
  • warfarin is an effective anti-coagulant in small, multiples doses.
  • One or two doses of the compound are seldom fatal if taken at the recommended concentration; thus the hazard of acute toxicity to man, domestic animal, and wildlife is greasy reduced.
  • the rodents begin to die after four or five daily doses of the materials, and the population is greatly reduced or eradicated in approximately three weeks. Death is caused by hemorrhages, brought about by the action of the warfarin in reducing the clotting power of the blood.
  • a method of determining a VKORC1 polypeptide sequence which conveys a coumarin effect exerted onto VKORC1 activity comprising the steps of:
  • VKORC1 polypeptide in another aspect of the present invention there is provided a VKORC1 polypeptide according to the invention, wherein the VKORC1 polypeptide contains at least one sequence abnormality, which exerts an effect on the activity of the VKORC1 polypeptide and wherein the VKORC1 polypeptide is the polypeptide according to SEQ ID No. 1 or 12 and the sequence abnormality is selected from the group consisting of V29L, V45A, R58G, R98W, L128R, and Y139C.
  • VKCFD2 multiple coagulation factor deficiency type 2
  • Fig. 1 depicts a comparison of the candidate interval of 3cM in the genetic map containing the VKORC1 gene locus in human, rat and mouse.
  • the ideogram of human chromosome 16 is shown, the area of homozygosity in the families 1 and 2 extends from 16p11.2 to 16q13 corresponding to approximately 25 Mb.
  • In the right part of the figure there are homologous parts of mouse and rat-chromosomes. Synthenic genes of 16p11.2 and 16q12.1 with homologous counterparts in Mus musculus (MMU) and Rattus norvegicus (RNO) are depicted.
  • MMU Mus musculus
  • RNO Rattus norvegicus
  • MMU Mus musculus
  • RNO Rattus norvegicus
  • PRKCB1 Rattus norvegicus
  • PRKCB1 Rattus norvegicus
  • IL4R Interleukin4 receptor á (human); II4ra: Interleukin4 receptor á (murin); II4r : Interleukin4 receptor á (rat);
  • SPS2 Selenophosphate synthetase (human); Sps2: Selenophosphate synthetase (murin/rat); HUMMLC2B :Myosin light Chain2 (human); Mylpf : Myosin light chain 2 (murin); Myl2 : Myosin light chaun 2 (rat);
  • SPN Sialophorine (human); Spn: Sialophorine (murin/rat).
  • Fig. 2 displays VKORC1 mutations in human vitamin K dependent clotting factor 2 (VKCFD2) and warfarin resistance (WR) patients.
  • VKCFD2 human vitamin K dependent clotting factor 2
  • WR warfarin resistance
  • the upper part of the figure shows the segregation of the R98W mutation in two VKCFD2 families and the electropherograms of a homozygous mutant (left) compared to a control (right).
  • the bottom part of the figure shows the heterozygous mutations of four WR patients. (85G>T, 134T>C, 172A>G, 383T>G) and a Rw rat (416A>G).
  • Fig. 3 shows a sequence alignment of VKORC1 and VKORC1 like protein 1 (VKORC1L1) polypeptides.
  • the alignment was generated with CLUSTALW and PRETTYBOX.
  • Human (hVKORC1), mouse (mVKORC1) and rat VKORC1 (rVKORC1) and VKORC1L1 polypeptides, i.e. VKORC1L1 of human (hVKORC1L1), mouse (mVKORC1L1) and Fugu rubripes (fVKORC1L1) share approximately 84% sequence identity within both groups and approximately 50% identity between both groups of proteins.
  • xVKORC1 depicts the VKORC1 polypeptide sequence of Xenopus laevis, fVKORC1 the VKORC1 polypeptide sequence of Fugu rubripes, and aVKORC1 the VKORC1 polypeptide sequence of Anopheles gambiae.
  • Tree analysis allows grouping the Fugu rubripes, Xenopus laevis and Anopheles gambiae proteins to the appropriate group.
  • the locations of the predicted transmembrane domains are underlined. Residues 29, 45, 58 and 128 mutated in WR patients are conserved in all species.
  • the arginine at position 98 mutated in the VKCFD2 patients is conserved in human, rat and mouse (plus sign).
  • Fig. 4 displays a northern blot analysis of VKORC1 in fetal and adult human tissues.
  • the upper blot depicts a northern blot of adult tissue, whereas the lower blot depicts a northern blot of fetal tissue.
  • Example 4 The lines with fragments of the sizes 2.4, 4.4, 7.5, UND 9.5 KB indicate molecular weight markers and allow estimation of the size of the all visible bands)
  • Fig. 5 shows the subcellular location of VKORC1.
  • VKORC1 constructs were stained with anti-calnexin (red; left column) and anti-GFP or anti-myc, respectively (green; middle column). Merged figures of the double-stained cells are shown in the right column.
  • Both VKORC1 constructs (tagged with GFP or myc) co-localize with the ER specific calnexin staining.
  • the control construct (pEGFP-N1) shows a diffuse staining pattern throughout the cytoplasm.
  • Fig. 6 displays a list of siRNA sequences for homo sapiens VKORC1 and primers endoding these siRNAs which can be used to express them using for example the siLentGeneTM U6 Cassette RNA Interference System.
  • Fig. 7 displays locations of siRNA targets in the coding sequence of homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1), which are shown in light grey; regions which are part of two possible siRNA targets are shown in darker grey; and regions with two or more possible siRNA sequences are shown in an even darker grey.
  • Hs_VKORC1 homo sapiens vitamin K epoxide reductase complex subunit 1
  • Fig. 8 provides a list of PCR primer sequences and PCR conditions for amplification of Homo sapiens VKORC1 and Homo sapiens VKORC1 L1.
  • Fig.9 provides a listing of the sequences their respective SEQ ID NOs.
  • FIG. 10 shows VKOR activities of HEK293 cells transfected with VKORC1 cDNA. Values are given as percent vitamin K epoxide converted into vitamin K quinone (product/residual substrate+product). Wildtype VKORC1 activity is also defined by being sensitive to warfarin (4.3 % residual activity at 80 ⁇ M warfarin compared to not inhibited). Mutations Y139C and V29L leading to resistance to warfarin exhibit 69 and 11 % residual activity at 80 ⁇ M warfarin respectively). All tests were run in duplicate. Untransfected and mock-transfected showed 1.49 and 0.96% activities, and were >90% inhibited by 10 ⁇ M warfarin. For further details see Example 7.
  • Fig. 11 shows the amino acid sequence of Homo sapiens vitamin K epoxide recycling polypeptide (HS_VKORC1; SEQ ID NO: 1)
  • Fig.12 shows the nucleic acid coding sequence of Homo sapiens vitamin K epoxide recycling polypeptide (HS_VKORC1; SEQ ID NO: 2)
  • Fig.13 shows the result of an ARMS-PCR experiment to determine whether or not a tested rat is warfarin resistant
  • Wildtype rats exhibited a band at 123 bp (probe# 3351, 3133, 3137, 3142, 4724, 4684, 3138, 3162), rats homozygous to the mutation (probe# 4701) exhibited a band at 101 bp and finally, rats with the heterozygous mutation (probe# 3066, 3350, 3352, 3354, 3139, 3140, 4754, 3146, 3148, 3149 ) showed two bands, one at 101 and another band at 123 bp.
  • Example 9 shows the result of an ARMS-PCR experiment to determine whether or not a tested rat is warfarin resistant
  • VKORC1 vitamin K epoxide recycling polypeptide
  • VKORC1 The VKORC1 gene was surprisingly identified in a mutant screen of warfarin resistant patients (for details cf. examples 1 and 2). There has been surprisingly identified a gene, VKORC1, which is mutated in patients with a combined deficiency of all vitamin K dependent coagulation factors (VKCFD2) and with warfarin resistance (WR), respectively, showing that VKORC1 polypeptide contains a binding site for warfarin and is a target of coumarin and its derivatives.
  • VKCFD2 vitamin K dependent coagulation factors
  • WR warfarin resistance
  • VKORC1 homologous genes are found in vertebrates (rat, mouse, Xenopus, Fugu), insects (Anopheles) and bacteria ( Fig. 3 ). Surprisingly, the three mammals and Fugu each have a second VKORC1-like gene of moderate similarity to the cognate gene. A number of amino acid positions within these genes are conserved throughout evolution.
  • VKORC1 A substitution of valine 29, arginine 58 leucine 128 - although dispersed over the entire VKORC1 polypeptide - obviously renders the inhibition of VKORC1 activity by warfarin ineffective. It can be speculated that these amino acids functionally co-operate in the tertiary structure of the VKORC1 protein 1. Taken together the mutation data in patients with two different phenotypes provide VKORC1 as the target protein, both for vitamin K and warfarin binding.
  • VKORC1 polypeptide refers to the full length sequence of the VKORC1 polypeptide comprising, preferably consisting of, a polypeptide sequence selected from the group consisting of:
  • the VKORC1 polypeptide is a target for coumarin and its derivatives in mammals.
  • VKORC1 polypeptide also encompasses Isolated VKORC1 polypeptides and VKORC1 polypeptides that are prepared by recombinant methods, e.g. by isolation and purification from a sample, from a host cell expressing the VKORC1 polypeptide, by screening a library and by protein synthesis, all of these methods being generally known to the person skilled in the art.
  • the entire VKORC1 polypeptide or parts thereof can be synthesized, for example, with the aid of the conventional synthesis such as the Merrifield technique.
  • VKORC1 polypeptide also encompasses polypeptides which have a sequence homology of about 80%, preferably about 90%, in particular, about 95%, especially about 98% with the VKORC1 polypeptide according to one of SEQ ID No. 1, 12, 17, 21, 25, and 27, provided that such VKORC1 polypeptide has VKORC1 activity.
  • VKORC1 polypeptide also encompasses homologous polypeptides which originate from organisms other than human, preferably from non-human mammals such as, rodents, e.g. mouse, rats, or monkeys and pigs and other vertebrates and invertebrates, such as those amino acid sequences according to SEQ ID, Nos.
  • VKORC1 polypeptide also includes VKORC1 polypeptides which are encoded by different alleles of the gene, in different individuals, in different organs of an organism or in different developmental phases, provided that such VKORC1 polypeptide has VKORC1 activity. It is further intended that the term "VKORC1 polypeptide” preferably also encompasses naturally occurring or synthetic mutations that exert no or only insignificant effects onto the activity of the VKORC1 polypeptide.
  • VKORC1 polypeptide preferably encompassed by the term "VKORC1 polypeptide” include VKORC1 polypeptides that may arise from differential splicing of the VKORC1 transcript, provided that such VKORC1 polypeptide has VKORC1 activity.
  • fragment of the polypeptide sequence is intended to encompass partial sequences of VKORC1 polypeptides, which fragments comprise, preferably consist of at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95% of the full length sequence of the VKORC1 polypeptide.
  • the fragment consists of a single contiguous sequence of the VKORC1 polypeptide but it may also contain at least two, at least three or at least about five different sequence portions of a VKORC1 polypeptide according to the invention which may or may not be interspaced by a heterologeous sequence or contain no extra polypeptide sequence at all.
  • sequence homology is understood as the degree of identity (% identity) of two sequences, that in the case of polypeptides can be determined by means of for example BlastP 2.0.1 and in the case of nucleic acids by means of for example BLASTN 2.014, wherein the Filter is set off and BLOSUM is 62 (Altschul et al., 1997).
  • VKORC1 activity within the meaning of the present invention is intended to mean the biological activity of the VKORC1 polypeptide of SEQ ID No.1. More preferably, “VKORC1 activity” is defined as the activity of the VKORC1 polypeptide to enzymatically convert (or support the enzymatic conversion) of vitamin K2,3-epoxide to vitamin K-quinone and/or the conversion of vitamin k quinone to vitamin K hydroquinone. VKORC1 activity may be determined using an assay based on the experiments described in detail in example 7 and figure 10 .
  • the VKORC1 polypeptides according to the present invention may be produced by a method described in more detail below.
  • the VKORC1 polypeptides are useful for identifying coumarin derivatives that avoid the problems described above.
  • they are useful for identifying coumarin derivatives, that effectively inhibit VKORC1 activity and that In independent assays are tested for (1) their metabolic half life in order to identify coumarin derivatives that are metabolized faster than the coumarins known In the art, (2) their ability to cause skin necrosis to identify coumarin derivatives that do not cause skin necrosis or to a lesser extend than the coumarins known in the art, (3) coumarin derivative-drug interactions in order to identify coumarin derivatives with lesser side effects than the coumarins known in the art.
  • the VKORC1 polypeptides according to the present invention are useful for identifying a VKORC1 sequence interacting with coumarin and its derivatives, and for treating patients having a decreased or increased VKORC1 activity relative to control levels.
  • VKORC1 nucleic acid relates to RNA or DNA, which may be a single or preferably a double stranded molecule.
  • the sequence of the VKORC1 nucleic acid may further comprise at least one intron and/or one polyA sequence.
  • VKORC1 nucleic acid may also encompass a precursor stage, for example a propolypeptide or prepropolypeptide, thereof. It is also understood that untranslated sequences can be present at the 5' end and/or the 3' end of the nucleic acid, without the activity of the encoded polypeptides being significantly altered. However, the DNA region encoding the VKORC1 polypeptide is particularly preferred.
  • VKORC1 nucleic acid may also encompass sequences which exhibit at least about 70%, in particular at least about 80%, especially at least about 90%, sequence homology with the sequence according to SEQ ID No.
  • the nucleic acid comprises a nucleic acid having a sequence complementary and/or antisense to a VKORC1 nucleic acid comprising, preferably consisting essentially of a nucleic acid sequence selected from the group consisting of:
  • the VKORC1 nucleic acid encodes a target for coumarin and its derivatives in mammals.
  • the VKORC1 nucleic acid may also comprises a non-functional mutant variant of the VKORC1 nucleic acid as defined above, such a variant containing a single nucleotide polymorphism (SNP) such as the nucleic acid sequences according to SEQ ID No. 8 and 9, provided that the VKORC1 polypeptide encoded by such nucleic acid has VKORC1 activity.
  • SNP single nucleotide polymorphism
  • stringent hybridization conditions is to be understood, in particular, as meaning those conditions In which a hybridization takes place, for example, at 60°C in 2.5x SSC buffer followed by several washing steps at 37°C in a lower buffer concentration and remains stable.
  • fragment of the nucleic acid sequence coding for a polypeptide having VKORC1 activity is understood to encompass nucleic acid sequence fragments comprising, preferably consisting of at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95% of the full length sequence coding for VKORC1 polypeptide according to the invention, preferably coding for the polypeptide according to SEQ ID No. 1, provided that the polypeptide encoded by such fragment has VKORC1 activity.
  • the fragment consists of a single contiguous sequence coding for the VKORC1 polypeptide but it may also contain at least two, at least three or at least about five different sequence portions, which may or may not be interspaced by a heterologeous sequence or contain no extra nucleic acid sequence at all, provided that all the sequence portions are arranged in the same reading frame. It is essential to the definition of these fragments that they display VKORC1 activity.
  • the VKORC1 nucleic acids can be produced by methods generally known to the skilled artisan. Nucleic acids may be prepared synthetically. Thus, the VKORC1 nucleic acids can, for example, be synthesized chemically, e.g. according to the phosphotriester method, with the aid of the DNA sequences as defined above and/or with the aid of the polypeptide sequences which are likewise defined above such as the SEQ ID No. 1 and by referring to the genetic code (see, e.g., Uhlmann, & Peyman, 1990). Preferably the VKORC1 nucleic acids are produced by recombinant gene technology methods generally known to the person skilled in the art.
  • VKORC1 nucleic acids are useful (1) for identifying coumarin derivatives that avoid the problems described above, (2) for producing PCR primers, DNA and RNA probes, siRNA or shRNA, and for VKORC1 polypeptide, (3) for treating patients having a decreased or increased VKORC1 activity relative to control values, and (4) for identifying coumarin derivatives that may be employed for pest control of rodents, all of which are described In detail below.
  • a host cell can be any host cell as defined below. Methods for selecting and culturing the host cells and for causing the host cells to express a polypeptide are generally known to the person skilled In the art. The same is true for methods of isolating the expressed polypeptide from the host cell; to this end an antibody according to the invention may be used for immunoaffinity precipitation.
  • the vector may contain a (poly)peptide tag that allows immunoaffinity precipitation by tag specific antibodies according to standard protocols known to the skilled worker (see also below).
  • polypeptide tag e.g. for the purpose of purifying the previously described VKORC1 polypeptides.
  • suitable protein tags enable the fusion proteins which are to be purified, to be absorbed with high affinity to a matrix. This is then followed, for example, by stringent washing with suitable buffers without eluting the fusion proteins to any significant extent, and, subsequently, specific elution of the fusion proteins.
  • protein tags which are known to the skilled person are a (His)6 tag, a Myc tag, a FLAG tag, a hemagglutinin tag, a glutathione transferase (GST) tag, intein having an affinity chitin-binding tag and a maltose-binding protein (MBP) tag.
  • GST glutathione transferase
  • MBP maltose-binding protein
  • VKORC1 nucleic acids may be introduced into a eukaryotic or prokaryotic cell by means of transfection, transformation or infection, and thereby enable the polypeptide to be expressed.
  • the VKORC1 nucleic acid can be present as a plasmid, or as a part of a viral or non-viral vector.
  • Particularly suitable viral vectors in this connection are: baculoviruses, vaccinia viruses, adenoviruses, adeno-assoclated viruses and herpes viruses.
  • Particularly suitable non-viral vectors are for example: virosomes, liposomes, cationic lipids and polylysine-conjugated DNA.
  • the vectors can be prokaryotic or eukaryotic expression vectors.
  • prokaryotic expression vectors are the pGEM vectors or pUC derivatives, which are used for expression in E . coli
  • eukaryotic expression vectors are the vectors p426Met25 or p426GAL1 (Mumberg et al., 1994) which are used for expression in Saccharamyces cerevisiae
  • Baculovirus vectors as disclosed in EP B1 0 127 839 or EP B1 0 549 721 , which are used for expression in insect cells
  • Rc/CMV and Rc/RSV, or SV40 vectors which are used for expression in mammalian cells, with all these vectors being generally available.
  • the expression vectors also contain promoters which are suitable for the respective cell, such as the trp promoter for expression in E. coli (see, e.g., EP-B1-0 154 133 ), the Met 25, GAL 1 or ADH2 promoter for expression in yeasts (Russel et al, 1983; Mumberg, see above), and the baculovirus polyhedrin promoter for expression in insect cells (see, e.g., EP B1 0127 839 ).
  • promoters which are suitable for the respective cell, such as the trp promoter for expression in E. coli (see, e.g., EP-B1-0 154 133 ), the Met 25, GAL 1 or ADH2 promoter for expression in yeasts (Russel et al, 1983; Mumberg, see above), and the baculovirus polyhedrin promoter for expression in insect cells (see, e.g., EP B1 0127 839 ).
  • Promoters which permit constitutive, regulatable, tissue-specific, cell type-specific, cell cycle-specific or metabolism-specific expression in eukaryotic cells are suitable, for example, for expression In mammalian cells.
  • Regulatable elements in accordance with the present invention are promoters, activator sequences, enhancers, silencers and/or repressor sequences.
  • Examples of preferred regulatable elements which permit constitutive expression in eukaryotes are promoters which are recognized by RNA polymerase III or viral promoters, CMV enhancer, CMV promoter, SV40 promoter or LTR promoters, e.g.
  • VKORC1 nucleic acids preferably takes place under the control of tissue-specific promoters.
  • the expression vectors may be used for preparing a VKORC1 polypeptides, DNA or RNA probes, or siRNA or shRNA, which can be used in accordance with the invention.
  • VKORC1 activity within the meaning of the invention relates to a level of activity and/or expression of the VKORC1 protein that is less than control level activity (as defined above) and/or expression determined in a healthy subject; the respective levels of activity may also be determined based on the assay as described in Example 7.
  • a host cell can be any cell suitable for expression of VKORC1 polypeptides and/or VKORC1 nucleic acids, preferably a HEK293-EBNA cell.
  • Cells can be either prokaryotic or eukaryotic cells, heterologeous or autologous cells.
  • prokaryotic cells are E. coll and examples of eukaryotic cells include primary hepatocytes cells, yeast cells, for example Saccharomyces cerevisiae or insect cells.
  • the host cell is a cell line, e.g. a COS-cell such as COS-7 cells or hepatocytes cell lines such as HepG2 cells.
  • the host cell Is preferably a non-human embryonic stem cell.
  • Methods for selecting and culturing host cells and for causing the host cells to express a polypeptide are generally known to the person skilled in the art. Processes for the transformation of cells and/or stem cells are likewise well known to a person skilled in the art and include, for example, electroporation or microinjection.
  • the host cells of the present invention can for example be employed for methods of identifying coumarin derivatives, for producing VKORC1 polypeptides and VKORC1 nucleic acids, siRNAs and shRNAs according to the invention, and for screening new drugs such as coumarin derivatives effecting VKORC1 activity and/or expression.
  • Transgenic animals in general show a tissue-specifically increased expression of the VKORC1 polypeptides and/or VKORC1 nucleic acids and can be used for the analysis of coagulation disorders and warfarin resistance and for development and evaluation of therapeutic strategies for such disorders.
  • Transgenic animals may further be employed in the production of VKORC1 polypeptide.
  • the polypeptide produced by the animal may for example be enriched in a body fluid of the animal.
  • transgenic animals in particular of transgenic mice, are likewise known to the person skilled in the art from DE 196 25 049 and US 4,736,866 ; US 5,625,122 ; US 5,698,765 ; US 5,583,278 and US 5,750,825 and include transgenic animals which can be produced, for example, by means of direct injection of expression vectors according to the present invention into embryos or spermatocytes or by injection of the expression vectors into the pronucleus of the fertilized ovum or by means of the transfection of expression vectors into embryonic stem cells or by nuclear transfer into appropriate recipient cells (Polites & Pinkert, 1994; Doetschman, in Pinkert, 1994, supra; Wood in Pinkert, 1994, supra ; Monastersky in Pinkert, 1994, supra ).
  • VKORC1 associated deficiency Is intended to encompass a disorder or disease that is associated with warfarin resistance, i.e. the patient displays a reduced or abolished susceptibility to treatment with coumarin or its derivatives, preferably the warfarin resistance results from a sequence abnormality of the VKORC1 polypeptide.
  • the term preferably also encompasses disorders or diseases associated with a level of activity and/or expression of VKORC1 that differs significantly from the condition in healthy patients, preferably the expression of VKORC1 polypeptide and/or its activity is significantly reduced or abolished which can e.g. be determined by measuring the prothrombin time, e.g. by international normalized ration (INR) protocol.
  • ITR international normalized ration
  • VKORC1 associated deficiency may be caused by a sequence abnormality in the VKORC1 polypeptide or VKORC1 nucleic acid as described in detail below. Moreover, when the level of expression and/or activity of VKORC1 polypeptide or VKORC1 nucleic acid is reduced or even completely abolished, gamma-carboxylation of vitamin K dependant proteins may also be impaired as well.
  • VKORC1 associated deficiency also encompasses diseases and/or disorder selected from familial multiple factor deficiency, a disorder or disease associated with decreased blood coagulation, such as hemophilia and a disorder associated decreased vascular calcification, diseases and/or disorders associated with impaired gamma-carboxylation of vitamin K dependant proteins.
  • VKORC1 polypeptide will be increased relative to the condition in healthy patients.
  • Such deficiencies which are also encompassed by the term the "VKORC1 associated deficiency" may be caused by a sequence abnormality in the VKORC1 polypeptide and/or the corresponding gene.
  • VKORC1 associated deficiency may be caused by a sequence abnormality in the VKORC1 polypeptide and/or the corresponding gene.
  • gamma-carboxylation of vitamin K dependant proteins may be facilitated as well.
  • VKORC1 associated deficiency may further comprise deficiencies selected from diseases or disorders associated with increased blood coagulation including patients suffering from a thrombus and/or patients having an increased risk of developing a thrombus, preferably due to a sequence abnormality in the VKORC1 polypeptide or its gene, diseases and/or disorders associated with improved gamma-carboxylation of vitamin K dependant proteins.
  • VKORC1 polypeptide and/or the corresponding gene may increase the susceptibility to treatment with coumarin and its derivatives in a patient having such sequence abnormality.
  • patients undergoing coumarin treating may show very low blood coagulation values.
  • Such disorders associated with increased susceptibility to treatment with coumarin are also intended to be encompassed by the term "VKORC1 associated deficiency".
  • Patients carrying a VKORC1 gene having a stop-mutation suffer from such deficiency associated with a increased coumarin sensitivity.
  • sample is intended to refer to a biomaterial comprising fetal or adult tissue or cell, preferably tissue or cells, preferably isolated or derived from heart, kidney and lung, pancreas, brain, placenta and skeletal muscle and blood, preferably from liver.
  • the sample can be isolated from a patient or another subject by means of methods including invasive or non-invasive methods. Invasive methods are generally known to the skilled artisan and comprise for example isolation of the sample by means of puncturing, surgical removal of the sample from the opened body or by means of endoscopic instruments. Minimally invasive and non-invasive methods are also known to the person skilled in the art and include for example, collecting body fluids such as blood, preferably by venopuncture, or urine or feces.
  • sample may also encompass a genomic or an expression library, preferably constructed based on an sample isolated from a patient, in which case techniques for isolation of the cDNA that are generally known to the skilled worker may be used.
  • the term "antibody” or “antibody fragment” is understood as also meaning antibodies or antigen-binding parts thereof prepared by genetic engineering and optionally modified, such as, for example, chimeric antibodies, humanized antibodies, multifunctional antibodies, bi- or oligospecific antibodies, single-stranded antibodies, F(ab) or F(ab)2 fragments (see, for example, EP-B1-0 368 684 , US 4,816,567 , US 4,816,397 , WO 88/01649 . WO 93/06213 , WO 98/24884 ).
  • the present invention provides a method of identifying a coumarin derivative which exerts an effect onto the activity of VKORC1 polypeptide as defined above, preferably a VKORC1 polypeptide comprising a polypeptide sequence selected from the group consisting of:
  • VKORC1 polypeptide is dithiothreitol-dependent conversion of vitamin K 2,3-epoxide to vitamin K quinone and wherein the significantly different activity value is a candidate activity value which is significantly higher than the control activity value, as described in further detail above and in Example 7 and Figure 10 . If essentially the same concentration of candidate coumarin derivative yields a lower percentage of vitamin K epoxide converted into vitamin K quinone (product/substrate+product) as warfarine does in this concentration, this is indicative ot the candidate coumarin derivative having a stronger inhibitory effect than warfarin, and vice versa.
  • control activity value is determined by a method comprising the steps of:
  • At least one additional compound is introduced into the host cell, which compound is selected from the group consisting of vitamin K, cytochrome B5, a nucleic acid coding for gamma-glutamyl-carboxylase, for microsomal epoxidehydrolase, for calumenin, or for glutathion-S-transferase.
  • a nucleic acid coding for gamma-glutamyl-carboxylase, for microsomal epoxidehydrolase, for calumenin, or for glutathion-S-transferase are selected from the group consisting of vitamin K, cytochrome B5, a nucleic acid coding for gamma-glutamyl-carboxylase, for microsomal epoxidehydrolase, for calumenin, or for glutathion-S-transferase.
  • Methods for introducing nucleic acids into host cells have been described in detail above.
  • the nucleic acids are expressed under
  • the methods of identifying a coumarin derivative are useful for developing novel coumarin derivatives that avoid at least one of the limitations of coumarin and its derivatives known in the art. If analysis of the kinetics of blood coagulation is included as a separate assay into the determination of VKORC1 polypeptide activity, the method according to the invention may be useful in identifying coumarin derivatives which mediate blood coagulation faster than coumarin and its derivatives known in the art and/or that are metabolized more rapidly so that accumulation of coumarin and its derivatives may be prevented or ameliorated and as a result the danger of overdosing is substantially decreased or even abolished.
  • such method of identifying may be combined with other assays such that coumarin derivatives may be identified which have a stronger (weaker) effect onto VKORC1 activity and thus in turn onto the blood coagulation and which coumarin derivatives in independent assays prove (1) to be metabolized more rapidly so that accumulation of coumarin may be prevented or ameliorated and as a result the danger of overdosing is substantially decreased or even abolished, (2) not to cause or to cause to lesser extend skin necrosis in patients or embryopathy if applied during pregnancy, and/or (3) to be metabolized faster or to be less more stabil and/or to be affected less by other drugs like Phenobarbital or amiodarone.
  • the assays which are suitable to screen for such properties of the coumarin derivatives and which are to be combined into a screen with the method of identifying a coumarin derivative according to the invention are generally known to the person skilled in the art.
  • coumarin is understood as meaning 3-(acetonylbenzyl)- 4-hydroxycoumarin, i.e. COUMADIN® or sodium warfarin.
  • derivative of coumarin is understood to encompass organic or inorganic compounds, peptides, polypeptides or complexes thereof, provided that they exert an effect onto the activity and/or expression of VKORC1 polypeptide, preferably an effect that inhibits the activity of the VKORC1 polypeptide, even more preferably a VKORC1 polypeptide-specific effect, i.e the coumarin derivative does not directly interact with other molecules involved in the coagulation pathway.
  • examples of such compounds are organic molecules that are derived from libraries of compounds, preferably those that have been analyzed for their pharmacological activity.
  • the derivatives of coumarin can influence the activity of the VKORC1 polypeptide in vivo and/or in vitro and enter into interactions of covalent or non-covalent manner with them.
  • the coumarin derivative is a chiral compound it is understood that "derivative of coumarin” also encompasses the respective R- and L-enantiomeril forms of the compound like those disclosed in WO 00/43003 .
  • the term “derivative of coumarin” refers to compounds derived from 4-hydroxycoumarin, especially compounds derived from COUMADIN. More preferably, “derivative of coumarin” also includes any coagulants which inhibits the regeneration of active vitamin K.
  • candidate coumarin derivative is understood to encompass organic or inorganic compounds, peptides, polypeptides or complexes. Examples of such compounds are organic molecules that are derived from libraries of compounds, preferably those that have been analyzed for their pharmacological activity. Preferably the term refers to compounds that are structurally related or derived from 4-hydroxycoumarin, especially compounds related or derived from COUMADIN. If the candidate coumarin derivative is a chiral compound it is understood that the respective R- and L-enantiomeric forms of the compound like those disclosed in WO 00/43003 are also encompassed by the term "candidate coumarin derivative".
  • the present invention provides a method of determining a VKORC1 polypeptide sequence which conveys a coumarin effect exerted onto VKORC1 activity, comprising the steps of:
  • control sequence activity value is determined by a method comprising the steps of:
  • the determined VKORC1 activity is dithiothreitol-dependent conversion of vitamin K 2,3-epoxide to vitamin K quinone and the significantly different value is a sequence abnormality activity value which is significantly higher than the control sequence activity value. Further details are provided above and in Example 7 and Figure 10 .
  • the method may be useful in identifying VKORC1 polypeptides that are less sensitive to coumarins. By introducing VKORC1 polypeptides with different sequence abnormalities this method allows Identification of sites of the polypeptide which are critical for the interaction of a tested coumarin and VKORC1. Such knowledge will for example be useful for designing new coumarins.
  • VKORC1 polypeptide sequence it is particularly preferred in the method of determining a VKORC1 polypeptide sequence that at least one additional compound is introduced into the cell which compound is selected from the group consisting of vitamin K, cytochrome B5, and a nucleic acid coding for gamma-glutamyl-carboxylase, for microsomal epoxidehydrolase, for calumenin, or for glutathion-S-transferase.
  • a further aspect of the present invention relates to a VKORC1 polypeptide wherein the VKORC1 polypeptide contains at least one sequence abnormality, which exerts an effect on the activity of the VKORC1 polypeptide and wherein the VKORC1 polypeptide is the polypeptide according to SEQ ID No. 1 or 12 and the sequence abnormality is selected from the group consisting of V29L, V45A, R58G, R98W, L128R and Y139C.
  • VKORC1 polypeptide containing at least one sequence abnormality can be generated by methods generally known to the skilled worker, including recombinant techniques and e.g. site directed mutagenesis, or by isolation the VKORC1 polypeptide having the at least one sequence abnormality from a sample obtained from a patient, preferably from a patient suffering from VKORC1 associated deficiencies. Methods for isolating proteins from a sample have been described in detail above.
  • VKORC1 polypeptide containing at least one sequence abnormality, which exerts an effect on the activity of the VKORC1 polypeptide can, for example, be used for generating antibodies binding specifically to these VKORC1 polypeptides. These antibodies in turn can be utilized for diagnosing VKORC1 associated deficiencies.
  • sequence abnormality is meant to encompass additions, insertions, deletions, substitutions of at least one amino acid that result in an alteration of the VKORC1 polypeptide sequence, preferably of the VKORC1 polypeptide sequence according to SE ID No. 1. Also encompassed are additions, insertions, deletions, substitutions of at least one nucleotide that lead to an altered amino acid sequence encoded by the VKORC1 nucleic acid sequence. Also encompassed are changes of the VKORC1 nucleic acid sequence that lead to a change in the reading frame of the nucleic acid sequence.
  • sequence abnormalities are indicated in the single letter amino acid code with the original amino acid being placed to the left of the number indicating the number of the amino acid In the polypeptide sequence according to SEQ ID No. 1.
  • the number to the right of the amino acid number indicates the amino acid that replaces the original amino acid.
  • the sequence abnormality V29L indicates that In position 29 of the VKORC1 polypeptide of SEQ ID No. 1 the amino acid valine (V) has been replaced by leucine (L).
  • the number in the code refers to the sequence position according to the numbering of the amino acids in that particular other polypeptide.
  • the invention provides a method of diagnosing a warfarin resistance or multiple coagulation factor deficiency type 2 (VKCFD2) in a patient comprising the steps of:
  • Methods for obtaining samples from a patient and for isolating total RNA or mRNA are generally known to the skilled worker, some of which have been described above.
  • Techniques for amplifying of DNA are not particularly limited and include PCR techniques which have also been described above.
  • techniques for reverse transcribing have been mentioned above and are not particularly limited and include reverse transcription using conventional protocols and commercially available kits that usually employ reverse transcriptase and oligo dT primers. The analysis may as well be based on genomic DNA isolated from a sample obtained from a patient.
  • the DNA is subjected to analysis in order to determine at least one sequence abnormality in a nucleic acid sequence coding for the VKORC1 polypeptide.
  • Methods for analyzing the amplified DNA are not particularly limited.
  • the amplified DNA is analyzed by a technique selected from the group consisting of PCR-based analysis, preferentially using PCR primers specific for the sequence abnormality, restriction digestion analysis, and DNA sequencing analysis.
  • the nucleic acid carrying the sequence abnormality is coding for a VKORC1 sequence having a sequence abnormality is selected from the group consisting of V29L(85 G>T), V45A(134 T>C), R58G (172 A>G), R98W (292 C>T), and L128R (383 T>G), Y139C (416 A>G).
  • the method of diagnosing the amplified DNA encodes at least a partial sequence of the VKORC1 polypeptide according to SEQ ID No. 1.
  • SEQ ID No. 1 One way of determining a sequence abnormality which is associated with a VKORC1 associated deficiency, is provided in Example 8.
  • VKORC1 sequences according to the invention containing the mutations 85 G>T, 134 T>C, 172 A>G, 292 C>T, and 383 T>G are provided in SEQ ID Nos. 3 to 7 and may be used as probes for diagnosing a VKORC1 associated deficiency using hybridization technique based analysis of nucleic acid samples obtained from a patient
  • VKORC1 expression may be detected on the level of the VKORC1 polypeptide. Therefore, in another aspect the present invention provides a method of diagnosing a warfarin resistance or multiple coagulation factor deficiency type 2 (VKCFD2) in a patient comprising the steps of:
  • Methods for detection of VKORC1 polypeptide having a sequence abnormality are not particularly limited, provided that the method allows specific detection of the protein carrying the sequence abnormality(ies). Examples of such methods preferably include immunohistochemical detection, immunoblotting, preferably Western blotting, and ELISA of the polypeptide, particularly preferred with antibodies specific for the VKORC1 polypeptide having a sequence abnormality as defined above. Analysis of sequence abnormalities on the level of the amino acid sequence is not particularly limited and may for example be carried out using VKORC1 antibodies which specifically recognize and bind a VKORC1 polypeptide having one or more sequence abnormalities.
  • the methods of diagnosing are used for diagnosing diseases and/or disorder selected from warfarin resistance, familial multiples factor deficiency, a disorder or disease associated with reduced or abolished blood coagulation, such as hemophilia and a disorder associated with decreased vascular calcification, diseases and/or disorders associated with impaired gamma-carboxylation of vitamin K dependant proteins.
  • diseases and/or disorder selected from warfarin resistance, familial multiples factor deficiency, a disorder or disease associated with reduced or abolished blood coagulation, such as hemophilia and a disorder associated with decreased vascular calcification, diseases and/or disorders associated with impaired gamma-carboxylation of vitamin K dependant proteins.
  • the methods of diagnosing may also be used to diagnose disorders and diseases associated with increased blood coagulation including patients and/or patients having an increased risk of developing a thrombus due to a sequence abnormality in the VKORC1 polypeptide or its gene, diseases and/or disorders associated with improved gamma-carboxylation of vitamin K dependant proteins.
  • the present invention provides a method of identifying a coumarin derivative which exerts an inhibitory effect onto the activity of a VKORC1 polypeptide having at least one sequence abnormality, comprising the steps of:
  • control sequence activity value is determined by a method comprising the steps of:
  • the activity of the VKORC1 polypeptide may be determined as described in detail above. And the method may be adopted from the method described in Example 7. Such method of identifying a coumarin derivative is useful for identifying coumarin derivatives that can be used as anticoagulants in warfarin resistance patients.
  • the sample can be any organ, tissue, body fluid or probe provided that it contains genomic DNA or mRNA from the rat to be tested.
  • the sample is blood, tissue from tail or ear urine or feces. Further details of the method are provided in Example 9.
  • Warfarin-resistant rodents have been described (Kohn & Pelz, 1999) and may be obtained from commercial suppliers (e.g. The Federal Biological Research Center for Agriculture and Forestry, Institute for Nematology and Vertebrate Research, Toppheideweg 88, 48161 Weg, Germany).
  • the warfarin-resistant rodent is a rodent transgenic for VKORC1 polypeptide containing at least one sequence abnormality, which sequence abnormality causes warfarin resistance. More preferred, the VKORC1 polypeptide containing at least one sequence abnormality is the polypeptide encoded by the nucleic acid sequence according to SEQ ID Nos.
  • sequence abnormality is selected from the group consisting of V29L (85 G>T), V45A(134 T>C), R58G (172 A>G), R98W (292 C>T), and L128R (383 T>G), Y139C (416 A>G).
  • the invention relates to the use of PCR primers according to SEQ ID No. 88 to 91 for determining whether or not a rat has a warfarin resistance genotype in a sample obtained from a rat.
  • coumarin and its derivatives are not particularly limited.
  • coumarin (warfarin) -containing toxicologically effective compositions are formulated as granular bait compositions containing from about 50-300 ppm, preferably about 250 ppm, of coumarin and its derivatives.
  • the bait is typically formulated with from 0.5% to 2.5% of warfarin concentrate in a suitable binder such as corn oil. If corn oil is used as a binder, it can be present in an amount of from about 0.5% to about 2% of the total composition.
  • the binder and warfarin are then mixed in e.g. with a product based on cereal products, corn meal, rolled oats, mixed animal feeds, and similar products known in the art.
  • the administration i.e.
  • the amount, formulation and frequency and duration of administration may follow standard protocols for assessing the toxicity of warfarin in rodents, more preferred follow standard protocols for assessing the lethal dose 50 (LD 50 ) value for a given poison to be tested, all of which are generally known to the skilled person and described in example 9
  • the coumarin derivative In order for the coumarin derivative to be toxicologically effective it is desirable that multiple ingestions are required to kill the rodent so that they do not develop bait shyness. Therefore, it is preferred to repeat the administration of the candidate coumarin derivative compositions a number of times. Usually the rodents begin to die after four or five daily doses of the compositions. Moreover, it may be preferred to suppress pain in the rats in order to ameliorate the suffering during the experiments by administration of pain suppressing agents generally known to the skilled worker. Inclusion of pain-suppressors in the coumarin and coumarin derivative composition may further be advantageous in order to further suppress the chance that rodents develop bait shyness.
  • the toxicity of the candidate coumarin derivative onto the rodent is determined which yields the candidate coumarin derivative toxicity value.
  • Methods for determining the toxicity of candidate coumarin derivatives are generally known to the skilled worker and include LD 50 analysis, analysis of the blood coagulation by determining the prothrombin time, e.g. by international normalized ration (INR) protocol.
  • the determined candidate coumarin derivative toxicity value is then compared with an appropriate control coumarin toxicity value determined on the basis of subjecting a different specimen of the warfarin-resistant rodent to the same treatment but exchanging the coumarin derivatives administration with an administration of a standard rodent coumarin compositions which the rodents are resistant for, commonly used for pest control.
  • the candidate coumarin derivative toxicity value is equal or preferably statistical significantly larger than the control coumarin toxicity value, the candidate coumarin derivative represents a toxicologically effective coumarin derivative.
  • VKCFD2 vitamin K-dependent clotting factor type 2
  • the human ortholog of Myl2, HUMMLC2B is located on chromosome 16p11 within the VKCFD2 candidate region and is part of a conserved linkage group of genes. Based on this synteny and on biochemical considerations, it is hypothesized that VKCFD2 and warfarin resistance may be due to allelic mutations in the same gene. If so, this would narrow down the critical interval in humans to a region of approximately 4.5 Mb between the interleukin 4 receptor gene (IL4R) and the integrin alpha M chain gene (ITGAM) on the short arm of chromosome 16 ( Fig. 1 ).
  • IGF interleukin 4 receptor gene
  • IGAM integrin alpha M chain gene
  • this region contains 141 Ensembl genes with approximately 1000 exons. Of these genes, 117 were annotated as known. Many of these genes could be excluded from further analysis because their function was well established and obviously not related to the metabolic steps of the vitamin K cycle. On the other side, genes upstream and downstream of this region are included that were regarded as functional candidates into the mutation screen.
  • VKCFD2 and WR subjects Fig. 2
  • This gene spans a genomic region of 5126 bp and comprises three exons coding for a protein of 163 amino acids. It was named vitamin K epoxide recycling protein 1 (VKORC1). Both non-related VKCFD2 patients and their affected siblings were found to harbor the same homozygous point mutation in the third exon (292C>T) whereas the parents were found to be heterozygous.
  • the mutation is caused by the replacement of arginine by tryptophan at amino acid residue 98 (R98W).
  • the families are of German and Lebanese origin.
  • the haplotypes in the region of homozygosity around the mutated gene were different in both families indicating independent mutation events.
  • three different heterozygous mutations were found leading to a valine by leucine substitution (patient C: V29L), an arginine by glycine substitution (patient D: R58G) and an exchange of leucine to arginine (patient E: L128R).
  • patient C valine by leucine substitution
  • patient D arginine by glycine substitution
  • patient E L128R
  • the R58G mutation is shared by the two affected brothers of index patient D.
  • the missense mutations were not present in 384 control chromosomes. Sequencing of the control chromosomes revealed two non-synonymous single nucleotide polymorphisms (C43C; L120L).
  • Genome sequences and annotation were obtained from NCBI, UCSC and Ensembl. Primers for mutation screening were designed using Primer3 software integrated into a script, ExonPrimer, to allow automatic primer design. For mutation screening, exons with intronic primers were amplified and amplified fragments were analyzed by direct sequencing with the BigDye Terminator Cycle sequencing kit (ABI)). Primer sequences were available on request. Topology predictions were performed using TMPRED and TMHM.
  • the PSORT II server predicted an ER membrane retention signal (KKXX or KXKXX) at position 159-163 of humans VKORC1 with a probability of 67 %[Jackson et al., 1990].
  • the consensus sequence was also present in the other VKORC1 proteins. This is in accordance with the likely location of the VKORC1 complex within the ER membrane system[Cain et al., 1997].
  • VKORC1 Tblastn searches with VKORC1 detected a homologous human (BC027734) and mouse gene (AK009497) showing 50% protein identity each. Both mRNA were wrongly predicted to code for proteins that show no homology with VKORC1. The predicted human protein starts at the third methionine. The mouse mRNA sequence is incomplete with a protein predicted in a different reading frame. The complete cDNA was established in mouse as well as in Fugu rubripes and partially in rat. These proteins were designated VKORC1 like protein 1 (VKORC1L1). Human, mouse and rat VKORC1L1 proteins share, approximately 84% identity between each other and approximately 50% identity with the corresponding VKORC1 proteins.
  • a homologous protein was further detected in Xenopus laevis (AAH43742) and - with weaker homology (1 e-14) - in Anopheles gambiae (EAA06271). Tree analysis suggested that both these proteins are orthologues to the VKORC1 gene.
  • VKORC1 seems to be widely expressed.
  • the corresponding Unigene entry contains more than 100 ESTs in various tissues.
  • the expression of VKORC1 in fetal and adult human tissues is examined by Northern blot analysis.
  • Human multiple tissue northern blots (Fetal Blot 1, Stratagene; Human 12-Lane, BD Clontech) contained 2 ⁇ g of poly(A)+-RNA.
  • Full-length human VKORC1 cDNA was radiolabeled using random primers DNA labeling system (Invitrogen life technologies) and hybridized using miracleHyb High-Performance Hybridization Solution (Stratagene).
  • a ⁇ -actin probe supplied with the multiple tissue northern blot was used for control hybridization.
  • VKORC1 expression levels can be observed in fetal and adult liver ( Fig. 4 ). High expression levels were also observed in fetal heart, kidney and lung, as well as in adult heart and pancreas. Fetal brain, adult placenta and skeletal muscle showed intermediate levels of expression. Minor expression levels were detected in adult brain, lung and kidney.
  • VKORC1-HindIII-F ATTAAGCTTCACCATGGGCAGCACCTGGGGGAGCCCT (SEQ ID No. 53)
  • VKORC1-EcoRI-R ATTGAATTCCGTGCCTCTTAGCCTTGCCCTG (SEQ ID No. 54).
  • the product was cloned into the pBluescript II vector (Stratagene) that was cleaved with the corresponding restriction enzymes and verified by direct sequencing.
  • the insert was re-cloned into the mammalian expression vectors pEGFP-N1 (BD Biosciences Clontech) and pcDNA3.1/myc-His (Invitrogen).
  • VKORC1 cDNA was cloned into the pcDNA3 vector (Invitrogen) after amplification with the primers VKORC1-pcdna3-F: GGGCGGAAGCTTGAGATAATGGGCA (SEQ ID No. 92) and VKORC1-pcdna3-R: GCTTGAATTCAGGGCTCAGTGC (SEQ ID No. 93). Mutagenesis was performed using the QuikChange mutagenesis Kit (Stratagene). Wildtype and mutated cDNAs were re-cloned for expression in pCEP4 (Invitrogen) using the Hin dIII and Xho I-sites. All constructs were verified by sequencing.
  • Example 6 Cell culture, transient transfection and immunocytochemistry and subcellular localization
  • VKORC1 activity purifies with the microsomal membrane fraction [Cain et al., 1997]. Furthermore, the gamma-glutamyl-carboxylase has been localized to the membrane of the endoplasmic reticulum by immunocytochemistry [Presnell, 2002 #31].
  • GFP- and myc-epitope tagged VKORC1 fusion protein constructs were generated for transient transfection experiments of COS-7 cells. Primary antibodies against the epitope tags and fluorochrome labeled secondary antibodies were used to visualize the fusion proteins. An antibody against the ER-specific protein calnexin served as a control.
  • COS-7 cells (DSMZ, Braunschweig) were maintained in Dulbecco's modified eagle's medium with 10% fetal calf serum.
  • Cells were plated on glass cover slips in six-well plates and after 18-24 h in culture transfected with the expression vector constructs using Effectene (QIAGEN) according to the manufacturer's specifications. After 48-60 h of further culturing, the cells were washed with PBS and fixed in 70% acetone / 30% methanol at -20 °C for 15 min. Following fixation, the cells were permeabilized in PBS, 0.1% Nonidet P-40 (SIGMA N-6507), and then blocked with PBS, 2% BSA and 0.1% NP-40 at 37°C.
  • SIGMA N-6507 Nonidet P-40
  • the green immunofluorescence of the VKORC1 fusion proteins decorated the mesh-like structures of the ER within the cytoplasm and perfectly co-localized with the label of the ER-marker calnexin (red) ( Fig. 5 ).
  • Example 7 An assay for determining enzymatic VKORC1 activity
  • Reaction was stopped by extraction of the substrate (Vitamin K2,3-epoxide) and the reaction products (Vitamin K-quinone and hydroquinone) using 1 ml 2-Propanol/Hexane (3:2, v/v); the organic supernatant was collected, dried and resolved in 50 ⁇ l methanol and analyzed with an HPLC at 254 nm.
  • the vitamin K quinone was separated from the epoxide by HPLC on a reversed phase C-18 column. During the extraction procedure vitamin K hydroquinone was quantitatively oxidized to the quinone form.
  • the output of the HPLC was analyzed automatically by calculating the area under the line of extinction of each peak.
  • the percentage of conversion of substrate was estimated by setting the area of the residual substrate-peak (epoxide) plus the product-peak (quinone) as 100 percent. Measurements were run in duplicate and the activity is given as percent of substrate converted into quinone.
  • Vitamin K 2,3-epoxide was prepared by oxidation of vitamin K quinone (Sigma-Aldrich) with H 2 O 2 . Warfarin (Sigma-Aldrich) was added in DMSO ( ⁇ 1 Vol%).
  • Example 8 A method of diagnosing a VKORC1 sequence abnormality
  • Genomic DNA of the specimen (human patient or mammal) is isolated according to standard procedures generally known to the skilled worker.
  • the genomic DNA of the desired Exon (1-3) of VKORC1 is amplified by PCR using specific primers which can also be designed by the skilled artisan.
  • the PCR-product is then purified using e.g. SAP/Exo (shrimp alkaline phosphatase and exonuclease) under standard conditions.
  • the purified DNA is then subjected to standard sequencing procedures such as: addition of 0.3 ⁇ l primer which is 10 pmol/ ⁇ l (forward or reverse primer) to 1 ⁇ l of the purified PCR-product; followed by addition of 8 ⁇ l DTCS-Mix (Beckman-Coulter) and 10.7 ⁇ l water; followed by cycle sequencing at First delay 96°C 60 sec Danaturation 95°C 30 sec Annealing Primerspecific(55-60°C) 30 sec Elongation 60°C 4 min
  • the probes are pipetted on a microtiter plate overlaid with a drop of paraffin-oil. Then separation in the sequencer at 4,2 V for 60-120 min follows.
  • the raw data is analyzed and the sequences are aligned with control sequences using the CEQ 2000 XL software (Version 4.3.9, Beckman Coulter). Differences between the control sequences (preferably the genomic VKORC1 nucleic acid sequence or its coding sequence according to SEQ ID NO. 2) and the sequenced DNA is indicative of the probes sequence to represent a VKORC1 nucleic acid containing a sequence abnormality.
  • Example 9 PCR-based assay for determining warfarin resistance in rats
  • rat (Rattus norvegicus) is warfarin resistant, i.e. whether the VKORC1 coding sequence according to SEQ ID No. 13 carries a mutation Y139C (416A>G)
  • the following assay based on ARMS-PCR was employed using rat feces as a source of rat genomic DNA.
  • rVKORC1-innerF one PCR primer that specifically hybridizes to the DNA sequence which contains the warfarin resistant mutated allele 416G
  • rVKORC1-innerR another PCR primer which specifically hybridizes to the Wildtype DNA sequence which contains the Wildtype allele 416A.
  • these two primers are oriented in opposite direction such that they pair with one out of two additional PCR primers included into the reaction.
  • the latter primers are located in different distances to and in opposite direction relative to the 416 site and as a result, depending on whether the 416 site is mutated or not either the rVKORC1-innerR primer or the rVKORC1-innerF primer will anneal and the PCR reaction will result in amplified DNA of a different size which is indicative of the genotype of the rat which DNA has been analyzed.
  • the PCR reaction will result in a band of 123 bp, whereas in rats homozygous to the mutation 416G the PCR reaction will yield a band at 101 bp.
  • the PCR reaction will give rise to two bands, one at 101 and another band at 123 bp.
  • the genomic DNA was isolated from the feces using standard DNA isolation procedures generally known to the skilled artisan. The following components were combined to a PCR reaction: 1 ⁇ l DNA (rat), 1 ⁇ l 5M Betain (Sigma), 2 pmol outer-Primer-F (1 ⁇ l of a 1:50-dilution), 2 pmol outer-Primer-R (1 ⁇ l of a 1:50-dilution), 10 pmol inner-Primer-F (1 ⁇ l of a 1:10-dilution), 10 pmol inner-Primer-R (1 ⁇ l of a 1:10- dilution), 0,25 ⁇ l Taq/Pfu-Polyme rase (1,25U Taq (Invitrogen) and 0,25 U Pfu (Stratagene)), ad 25 ⁇ l PCR-buffer (1 ml PCR-buffer contains: 100 ⁇ l 10xPCR-buffer (Invitrogen), 160 ⁇ l nucleotide stem-solution (1,25 mM
  • the PCR conditions were: 95°C for 3 min, followed by 32 cycles of: 95°C for 20 sec, 62°C for 20 sec, and 70°C for 10 sec. Finally, the reaction is incubated at 70°C for 3 min.
  • the PCR Products were separated by gel electrophoresis on a 3,5 % TAE-Agarose-Gel with ethidium bromide (10 ⁇ l of a 1 %-stem solution for every 100ml). The gelelectrophosesis was allowed to run for 30 min at 130 V.
  • Rats (Rattus norvegicus) with and without warfarin resistance were used for the assay.
  • the results of the PCR are shown in Figure 13 . Wildtype rats exhibited a band at 123 bp, rats homozygous to the mutation exhibited a band at 101 bp and finally, rats with the heterozygous mutation showed two bands, one at 101 and another band at 123 bp.
  • this assay can be employed to determine whether a given rat is warfarin resistant or not.
  • Such assays are highly versatile in order to manage pest control in a given region, since knowledge of the frequency of warfarin resistant rats is critical for deciding which pesticide may be employed effectively. If in a given region there is a high prevalence of warfarin resistant rats warfarin and analogues thereof are an unsuitable means to kill the rats. If, however, the determined frequency of warfarin resistant rats is low, warfarin may be effectively used to fight rodents.

Abstract

The present invention relates to a method for gamma-carboxylating a vitamin K-dependent polypeptide in a host cell comprising introducing into said host cell a nucleic acid that encodes VKORC1 in a manner that the nucleic acid encoding VKORC1 is recombinantly expressed in said cell. Further, the present invention relates to a use of a recombinant VKORC1 nucleic acid for gamma-carboxylating a vitamin K-dependent polypeptide in a host cell.

Description

    Field of the Invention
  • The invention relates to methods for identifying coumarin derivatives, and also claims VKORC1 polypeptides and VKORC1 nucleic acids containing a sequence abnormality associated with a VKORC1 associated deficiency such as warfarin resistance, wherein the VKORC1 polypeptides and VKORC1 nucleic acids can be used for diagnosing these deficiencies. Moreover, the invention relates to methods for identifying coumarin derivatives usable In pest control of rodents.
    • Background of the invention
  • Repression of untimely blood coagulation is the therapeutic option of choice for acute treatment and long-term prevention of thrombolic events. Among the anti-coagulants coumarin are widely used for the prevention of thrombosis such as in patients Immobilized after surgery, patients having a chronic heart failure, patients having atherosclerotic vascular disease, patients having a malignancy, and patients that are pregnant. Moreover, coumarins are the most widely used oral anticoagulants for the treatment and prophylaxis of thrombosis [Suttie, 1987]. Coumarins are typically derivatives of 6-hydroxycoumarin, such as 3-(acetonylbenzyl)-4- hydroxycoumarin (COUMADIN®).
  • The coumarins target the blood coagulation cascade indirectly by inhibition of the vitamin K cycle.
  • Vitamin K is an essential cofactor for the post-translational activation by gamma-carboxylation of a group of regulatory proteins, the Gla-proteins. In several metabolic pathways, some key proteins require carboxylation for proper function. The blood coagulation cascade is the best-studied example. Here, the procoagulant factors II, VII, IX and X, and the anticoagulant factors protein C, protein S, protein Z are dependent on gamma-carboxylation. This post-translational modification enables the attachment of the modified proteins - in the presence of calcium - to phospholipid- bilayer membranes which is an essential step in the activation of blood coagulation [Sperling et al., 1978][Esmon et al., 1975]. Other proteins requiring gamma-carboxylation are the matrix gla protein and osteocalcin, both regulators of bone metabolism[Price, 1988] and the "growth arrest specific gene", a signal transduction protein of the cell cycle[Manfioletti et al., 1993][Stitt et al., 1995].
  • During gamma-carboxylation, a carboxyl group is introduced into glutamate residues of the target proteins by the enzyme gamma-glutamyl carboxylase (GGCX) in liver microsomes [Furie & Furie, 1988][Suttie, 1987]. The reaction requires as a cofactor stoichiometric amounts of reduced vitamin K1 hydroquinone (vitamin K1H2) which is oxidized to vitamin K-2,3 epoxide[Cain et al., 1997]. The regeneration of the active cofactor is mediated by a multi-protein complex termed vitamin K-2,3-epoxide reductase (VKOR) [Wallin & Martin, 1985]. The same complex is targeted by the coumarin-type poisons used in rodent pest control. This "vitamin K cycle" has been characterized biochemically in great detail but the molecular components have not yet been purified to homogeneity [Guenthner et al., 19981]. Moreover, the molecular nature of coumarin activity and the molecules interacting with coumarins are still elusive.
  • It is generally appreciated in the art that although largely effective, there are a number of limitations to the use of coumarins. First of all; there are humans that are inert to coumarin treatment. The term warfarin resistance (WR) is used for individuals who maintain normal clotting factor activities despite oral anticoagulation by coumarins (OMIM Access. No. 122700). Autosomal dominant transmission has been observed in several pedigrees [O'Reilly et al., 1964][O'Reilly, 1970]. Combined deficiency of all vitamin K dependent clotting factors (VKCFD) is a very rare bleeding disorder in humans of autosomal recessive inheritance with 14 cases described as yet[McMillan & Roberts, 1966][Fischer, 1966][Johnson et al., 1980][Goldsmith et al., 1982][Vicente et al., 1984][Ekelund et al., 1986][Pauli et al., 1987][Leonard, 1988][Pechlaner et al., 1992][Boneh & Bar-Ziv, 1996][Brenner et al., 1998][Spronk et al., 2000][Oldenburg et al., 2000]. Clinical symptoms of the disease include episodes of perinatal intracerebral hemorrhage sometimes with fatal outcome. The bleeding tendency is usually completely reversed by oral administration of vitamin K. Additional symptoms in newborns can resemble warfarin embryopathy with nasal and distal phalangeal hypoplasia and premature calcification of epiphyses[Pauli et al., 1987]. The disease may result either from a defective resorption/transport of vitamin K to the liver [Prentice, 1985] or from mutations in one of the genes involved in gamma-carboxylation. In subtype 1 (VKCFD1, OMIM # 277450), mutations in the GGCX gene on chromosome 2p12 result in insufficient carboxylation of clotting factors [Brenner et al., 1998][Spronk et al., 2000]. There has been described a linkage of two kindreds with familial multiple coagulation factor deficiency (FMFD, now re-named: VKCFD2, OMIM # 607473) to a 20 Mb interval of the pericentric region of chromosome 16p12-q21 [Fregin et al., 2002]. Patients with VKCFD2 showed significantly increased serum levels of vitamin K epoxide, thus suggesting a defect in one of the subunits of the VKOR complex. Taken together, there is evidence that there are patients that display warfarin resistance. As a result, there is a need to identify novel coumarins derivatives that are effective anticoagulants for treating these patients, and methods for identifying these coumarin derivatives.
  • The use of coumarins is associated with a risk of spontaneous bleedings, with a significant mortality rate. Moreover, the prediction of the accurate coumarin maintenance dose is difficult. In the absence of the target molecule which coumarin exerts an effect on, the treatment regimen has to be established, on a patient-by-patient basis. During the time the optimum regimen is yet not established the patient either suffers from an increased risk of thrombogenesis or of an increased risk of bleeding. Therefore there is a need for a method of determining the optimal treatment regimen that is faster and saver. Further, establishing an optimal treatment regimen is complicated by the fact, that there is a considerable delay between the administration of coumarins and the onset of its anticoagulant activity. Given the delayed action of coumarin and given the fact that coumarin tends to accumulate in time there is a need for coumarin derivatives that effect blood coagulation faster than the coumarins known in the art. By the same token there is also a need for coumarins that are metabolized more rapidly so that accumulation of coumarin may be prevented or ameliorated and as a result the danger of overdosing is decreased or abolished.
  • It is well appreciated that if coumarin treatment is initiated during a thrombic state, the levels of protein C and S decline, thus temporarily creating a thrombogenic potential which is usually compensated for by overlapping heparin and coumarin administration for a number of days. Again, there is a need to identify the molecular target of coumarin action in order to be able to screen for novel coumarin derivatives that do not possess these limitations or at least to a lesser extend.
  • A coumarin therapy sometimes induces skin necrosis in patients and if applied during pregnancy may cause embryopathy creating a need for novel coumarin derivatives which do not cause these effects.
  • There are a number of interactions between drugs and coumarins. Some of these drugs like Phenobarbital induce lower plasma levels of coumarins due to an increased metabolization of coumarin which is believed to be caused by the mixed-function oxidases like the cytochrome P450 mixed-function oxidases. Such interaction is of clinical relevance if the appropriate regimen of e.g. Phenobarbital and coumarin has been determined and later on only administration of Phenobarbital is discontinued leading to a rise of the plasma level of coumarin which causing excessive anticoagulation. Other drugs like Amiodarone cause a delayed metabolization of coumarin leading again to excessive anticoagulation if co-administered with coumarins. Since the molecules affected by coumarins are not known in the art there is a need to develop novel coumarins and tools to identify the latter in order to solve these problems.
  • WO 00/03015 describes nucleic acid and amino acid sequences of human transport protein homologs and the use thereof (cf. for examples page 1, lines 5 to 7). Further, the Database EMBL, Mus musculus cDNA library (XP002318821) describes the cDNA of a Mus musculus 13 days embryo head (cf. for example the title). Additionally, Fregin, A. et al, Blood, Vol. 100, No. 9, 1 November 2002, describes the assignment of a second gene for hereditary combined multiple coagulation factor deficiency to chromosome 16 (cf. for example page 3231, left hand column, lines 22 to 25) and Zimmermann, A. et al., Biochemical Pharmacology, Vol. 23, pp. 1033-1040, describes the influence of warfarin on prothrombin formation and the regeneration of vitamin K from vitamin K-2,3-epoxide (cf. for example the abstract).
  • Finally, coumarins, especially warfarin, are not only used in humans but since the 1950s, coumarins have been in use as an active ingredient in rodenticidal compositions. The basis for the effectiveness of warfarin as a rodenticide lies in the fact that it is an effective anti-coagulant in small, multiples doses. One or two doses of the compound are seldom fatal if taken at the recommended concentration; thus the hazard of acute toxicity to man, domestic animal, and wildlife is greasy reduced. Usually the rodents begin to die after four or five daily doses of the materials, and the population is greatly reduced or eradicated in approximately three weeks. Death is caused by hemorrhages, brought about by the action of the warfarin in reducing the clotting power of the blood. These hemorrhages may be external or internal and can be initiated by very slight injury or capillary damage. One of the other advantages of coumarins is that, because multiple ingestions are required to kill the rodents, they do not develop bait shyness. Beginning in 1969, rodents - particularly rats and, to a somewhat lesser extent, mice - began showing resistance to warfarin baits. The general assumption was that such resistance had a genetic basis. As for the mechanism, it is the VKORC1 complex mentioned above that is targeted by derivatives of warfarin in use for rodent pest control. [Jackson et al., 1988]. Resistance to coumarin derivatives has arisen spontaneously, in several wild rodent populations rendering the use of these drugs locally Ineffective for pest control. Autosomal dominant loci for warfarin resistance have been mapped in the mouse (War) to chromosome 7 [Wallace et al., 1976] and In rat (Rw) to the long arm of chromosome 1[Greavses & Ayres, 1967][Kohn & Pelz, 1999]. Since the VKOR complex is the target of the coumarin drugs resistance is thought to be mediated by alterations in one of its protein components [Jackson, 1988]. The development of resistance in rodents has created a need for identifying the target of coumarins action which would facilitate the development of novel coumarin-derivatives for use in pest control.
  • Taken together it is an object of the present invention to provide a target molecule for coumarin and its derivatives In mammals. It is another object of the present invention to provide methods for identifying novel coumarins which solve at least one of the problems mentioned above. It Is a further object of the present invention to identify polypeptides and nucleic acids coding for them which cause warfarin resistance in human and non-human mammals, preferably rodents. It is also an object of the present invention to diagnose, prevent and/or treat disorders and diseases selected from diseases from warfarin resistance, familial multiple factor deficiency, a disorder or disease associated with increased blood coagulation such as patients suffering from a thrombus and/or patients having an increased risk of developing a thrombus, such as an inherited increased risk of thrombogenesis, preferably an increased risk of thrombogenesis due to a surgery or due to pregnancy, and increased vascular calcification. Moreover, it is also an object of the present invention to diagnose, prevent and/or treat diseases or disorders associated with attenuated blood coagulation, such as hemophilia, disorder associated decreased vascular calcification and disorders and diseases with an increased risk of bleeding. Finally, it is an object of the present Invention to provide a method for identifying coumarin and its derivatives which are effective in pest control of non-human mammal and compositions for killing rodents.
    • Summary of the Invention
  • In solving the above objects there is provided a method of identifying a coumarin derivative which exerts an effect onto the activity of VKORC1 polypeptide comprising a polypeptide sequence selected from the group consisting of:
    1. (a) a polypeptide sequence selected from the group consisting of a sequence according to SEQ ID No. 1, 12, and 17;
    2. (b) a polypeptide sequence of an allele of the polypeptide sequence defined in (a);
    3. (c) a polypeptide sequence having at least 80% homology with the polypeptide sequence defined in (a) or (b), wherein the polypeptide sequence has VKORC1 activity; and
    4. (d) a polypeptide sequence of a fragment of the polypeptide sequence defined in (a), (b) or (c) having VKORC1 activity,
    comprising the steps of:
    • (I) providing a host cell having been introduced the VKORC1 nucleic acid or a vector containing the VKORC1 nucleic acid;
    • (II) expressing the VKORC1 polypeptide in the host cell;
    • (III) administering a candidate coumarin derivative;
    • (IV) determining the activity of VKORC1 polypeptide (candidate activity value);
    • (V) comparing the candidate activity value with a control activity value; and
    • (VI) identifying the candidate coumarin derivative as a coumarin derivative exerting an effect onto the activity of the VKORC1 polypeptide, provided the candidate activity value is significantly different from the control activity value.
  • Furthermore, according to another aspect of the present invention there is provided a method of determining a VKORC1 polypeptide sequence which conveys a coumarin effect exerted onto VKORC1 activity, comprising the steps of:
    • (I) providing a cell expressing the VKORC1 polypeptide according to the Invention, which VKORC1 polypeptide has at least one sequence abnormality;
    • (II) administering coumarin or a derivative thereof to the cell;
    • (III) determining the activity of the VKORC1 polypeptide (sequence abnormality activity value); and
    • (IV) comparing the sequence abnormality activity value with the control sequence activity value,
    wherein a significant deviation of the sequence abnormality activity value from to the control sequence activity value is indicative that the sequence abnormality of the VKORC1 polypeptide conveys the coumarin effect exerted onto VKORC1 polypeptide.
  • In another aspect of the present invention there is provided a VKORC1 polypeptide according to the invention, wherein the VKORC1 polypeptide contains at least one sequence abnormality, which exerts an effect on the activity of the VKORC1 polypeptide and wherein the VKORC1 polypeptide is the polypeptide according to SEQ ID No. 1 or 12 and the sequence abnormality is selected from the group consisting of V29L, V45A, R58G, R98W, L128R, and Y139C.
  • Moreover, according to another aspect of the present invention there is provided a method of diagnosing a warfarin resistance or multiple coagulation factor deficiency type 2 (VKCFD2) in a patient comprising the steps of:
    • (I) amplifying a DNA sample obtained from the patient or reverse transcribing a RNA sample obtained from the patient into a DNA and amplifying the DNA; and
    • (II) analyzing the amplified DNA of step (I) to determine at least one sequence abnormality in a nucleic acid sequence cording for a VKORC1 polypeptide of or in an amino acid sequence of a VKORC1 polypeptide;
    wherein the determined sequence abnormality is indicative of the patient suffering from a VKORC1 associated deficiency; preferably the sequence abnormality exerts an effect on the activity of the VKORC1 polypeptide.
  • In addition, according to another aspect of the present invention there is provided a method of Identifying a coumarin derivative which is toxicologically effective in warfarin-resistant rodents comprising the steps of:
    • (I) providing a warfarin-resistant rodent;
    • (II) administering a candidate coumarin derivative to the rodent,
    • (III) determining the toxicity of the candidate coumarin derivative onto the rodent (candidate coumarin derivative toxicity value);
    • (IV) comparing the candidate coumarin derivative toxicity value with a control coumarin toxicity value;
    • (V) identifying the candidate coumarin derivative as a rodenticidally effective coumarin derivative provided that the candidate coumarin derivative toxicity value is significantly larger than the control coumarin toxicity value.
    Brief description of the drawings
  • Fig. 1 depicts a comparison of the candidate interval of 3cM in the genetic map containing the VKORC1 gene locus in human, rat and mouse. The ideogram of human chromosome 16 is shown, the area of homozygosity in the families 1 and 2 extends from 16p11.2 to 16q13 corresponding to approximately 25 Mb. In the right part of the figure there are homologous parts of mouse and rat-chromosomes. Synthenic genes of 16p11.2 and 16q12.1 with homologous counterparts in Mus musculus (MMU) and Rattus norvegicus (RNO) are depicted. The loci for phenotype resistance to warfarin in the mouse (War) and rat (Rw) are mapped to regions homologous to 16p11.2. MMU: Mus musculus; RNO: Rattus norvegicus; PRKCB1; Prkcb; IL4R : Interleukin4 receptor á (human); II4ra: Interleukin4 receptor á (murin); II4r : Interleukin4 receptor á (rat); SPS2: Selenophosphate synthetase (human); Sps2: Selenophosphate synthetase (murin/rat); HUMMLC2B :Myosin light Chain2 (human); Mylpf : Myosin light chain 2 (murin); Myl2 : Myosin light chaun 2 (rat); SPN: Sialophorine (human); Spn: Sialophorine (murin/rat).
  • Fig. 2 displays VKORC1 mutations in human vitamin K dependent clotting factor 2 (VKCFD2) and warfarin resistance (WR) patients. The upper part of the figure shows the segregation of the R98W mutation in two VKCFD2 families and the electropherograms of a homozygous mutant (left) compared to a control (right). The bottom part of the figure shows the heterozygous mutations of four WR patients. (85G>T, 134T>C, 172A>G, 383T>G) and a Rw rat (416A>G).
  • Fig. 3 shows a sequence alignment of VKORC1 and VKORC1 like protein 1 (VKORC1L1) polypeptides. The alignment was generated with CLUSTALW and PRETTYBOX. Human (hVKORC1), mouse (mVKORC1) and rat VKORC1 (rVKORC1) and VKORC1L1 polypeptides, i.e. VKORC1L1 of human (hVKORC1L1), mouse (mVKORC1L1) and Fugu rubripes (fVKORC1L1), share approximately 84% sequence identity within both groups and approximately 50% identity between both groups of proteins. xVKORC1 depicts the VKORC1 polypeptide sequence of Xenopus laevis, fVKORC1 the VKORC1 polypeptide sequence of Fugu rubripes, and aVKORC1 the VKORC1 polypeptide sequence of Anopheles gambiae. Tree analysis allows grouping the Fugu rubripes, Xenopus laevis and Anopheles gambiae proteins to the appropriate group. The locations of the predicted transmembrane domains are underlined. Residues 29, 45, 58 and 128 mutated in WR patients are conserved in all species. The arginine at position 98 mutated in the VKCFD2 patients is conserved in human, rat and mouse (plus sign).
  • Fig. 4 displays a northern blot analysis of VKORC1 in fetal and adult human tissues. The upper blot depicts a northern blot of adult tissue, whereas the lower blot depicts a northern blot of fetal tissue. For more details see Example 4. The lines with fragments of the sizes 2.4, 4.4, 7.5, UND 9.5 KB indicate molecular weight markers and allow estimation of the size of the all visible bands)
  • Fig. 5 shows the subcellular location of VKORC1. For more details see example 6. To this end, COS-7 cells transiently transfected with VKORC1 constructs were stained with anti-calnexin (red; left column) and anti-GFP or anti-myc, respectively (green; middle column). Merged figures of the double-stained cells are shown in the right column. Both VKORC1 constructs (tagged with GFP or myc) co-localize with the ER specific calnexin staining. The control construct (pEGFP-N1) shows a diffuse staining pattern throughout the cytoplasm.
  • Fig. 6 displays a list of siRNA sequences for homo sapiens VKORC1 and primers endoding these siRNAs which can be used to express them using for example the siLentGene™ U6 Cassette RNA Interference System.
  • Fig. 7 displays locations of siRNA targets in the coding sequence of homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1), which are shown in light grey; regions which are part of two possible siRNA targets are shown in darker grey; and regions with two or more possible siRNA sequences are shown in an even darker grey.
  • Fig. 8 provides a list of PCR primer sequences and PCR conditions for amplification of Homo sapiens VKORC1 and Homo sapiens VKORC1 L1.
  • Fig.9 provides a listing of the sequences their respective SEQ ID NOs.
  • Fig. 10 shows VKOR activities of HEK293 cells transfected with VKORC1 cDNA. Values are given as percent vitamin K epoxide converted into vitamin K quinone (product/residual substrate+product). Wildtype VKORC1 activity is also defined by being sensitive to warfarin (4.3 % residual activity at 80µM warfarin compared to not inhibited). Mutations Y139C and V29L leading to resistance to warfarin exhibit 69 and 11 % residual activity at 80 µM warfarin respectively). All tests were run in duplicate. Untransfected and mock-transfected showed 1.49 and 0.96% activities, and were >90% inhibited by 10 µM warfarin. For further details see Example 7.
  • Fig. 11 shows the amino acid sequence of Homo sapiens vitamin K epoxide recycling polypeptide (HS_VKORC1; SEQ ID NO: 1)
  • Fig.12 shows the nucleic acid coding sequence of Homo sapiens vitamin K epoxide recycling polypeptide (HS_VKORC1; SEQ ID NO: 2)
  • Fig.13 shows the result of an ARMS-PCR experiment to determine whether or not a tested rat is warfarin resistant Wildtype rats exhibited a band at 123 bp ( probe# 3351, 3133, 3137, 3142, 4724, 4684, 3138, 3162), rats homozygous to the mutation (probe# 4701) exhibited a band at 101 bp and finally, rats with the heterozygous mutation ( probe# 3066, 3350, 3352, 3354, 3139, 3140, 4754, 3146, 3148, 3149 ) showed two bands, one at 101 and another band at 123 bp. For further details see Example 9.
    • Detailed description of the Invention
  • In order to meet the needs for developing novel coumarin derivatives and for identifying the target of coumarin and its derivatives the vitamin K epoxide recycling polypeptide (VKORC1) was cloned. This gene was previously unknown spanning a genomic region of 5126 bp and comprising three exons coding for a protein of 163 amino acids. Topology analysis suggests a highly hydrophobic protein with at least two transmembrane domains. This is compatible with the known location of the VKORC1 complex activity in ER membranes and with immunofluorescence data in COS-7 cells transfected with VKORC1 constructs (Fig. 5).
  • The VKORC1 gene was surprisingly identified in a mutant screen of warfarin resistant patients (for details cf. examples 1 and 2). There has been surprisingly identified a gene, VKORC1, which is mutated in patients with a combined deficiency of all vitamin K dependent coagulation factors (VKCFD2) and with warfarin resistance (WR), respectively, showing that VKORC1 polypeptide contains a binding site for warfarin and is a target of coumarin and its derivatives. The evidence that the mutations are causative of the two phenotypes is as follows:
    1. (i) an R98W mutation segregates with the disease in two unrelated families with VKCFD2;
    2. (ii) this arginine at position 98 is conserved in the human and in the homologous mouse and rat genes, respectively,
    3. (iii) three warfarin resistant brothers share an R58G substitution;
    4. (iv) this amino acid and the other residues found mutated In two more unrelated WR patients (V29L and L128R) are conserved in all species analyzed except for three bacterial genes (see Fig. 3); and
    5. (v) none of the 5 presumed mutations was found in 192 control DNA samples.
  • Moreover, homology searches in genome and protein databases have not revealed any similarities of VKORC1 to any protein or peptide domain of annotated function. However, homologous genes are found in vertebrates (rat, mouse, Xenopus, Fugu), insects (Anopheles) and bacteria (Fig. 3). Surprisingly, the three mammals and Fugu each have a second VKORC1-like gene of moderate similarity to the cognate gene. A number of amino acid positions within these genes are conserved throughout evolution. This is in accordance with the well established fact that gamma-carboxylation - and thus the use of vitamin K as a cofactor of this process - is an evolutionary old post-translational protein modification [Bandyopadhyay et al., 2002].
  • A substitution of valine 29, arginine 58 leucine 128 - although dispersed over the entire VKORC1 polypeptide - obviously renders the inhibition of VKORC1 activity by warfarin ineffective. It can be speculated that these amino acids functionally co-operate in the tertiary structure of the VKORC1 protein 1. Taken together the mutation data in patients with two different phenotypes provide VKORC1 as the target protein, both for vitamin K and warfarin binding.
  • Within the meaning of the invention the term "VKORC1 polypeptide refers to the full length sequence of the VKORC1 polypeptide comprising, preferably consisting of, a polypeptide sequence selected from the group consisting of:
    1. (a) a polypeptide sequence selected from the group consisting of a sequence according to SEQ ID No. 1,12,17, 21, 25, and 27;
    2. (b) a polypeptide sequence of an allele of the polypeptide sequence defined in (a);
    3. (c) a polypeptide sequence having at least 80% homology with the polypeptide sequence defined in (a) or (b), which polypeptide sequence has VKORC1 activity; and
    4. (d) a polypeptide sequence of a fragment of the polypeptide sequence defined in (a), (b) or (c) having VKORC1 activity.
  • Preferably, the VKORC1 polypeptide is a target for coumarin and its derivatives in mammals.The term "VKORC1 polypeptide" also encompasses Isolated VKORC1 polypeptides and VKORC1 polypeptides that are prepared by recombinant methods, e.g. by isolation and purification from a sample, from a host cell expressing the VKORC1 polypeptide, by screening a library and by protein synthesis, all of these methods being generally known to the person skilled in the art. Preferably, the entire VKORC1 polypeptide or parts thereof can be synthesized, for example, with the aid of the conventional synthesis such as the Merrifield technique. More preferably, the term "VKORC1 polypeptide" also encompasses polypeptides which have a sequence homology of about 80%, preferably about 90%, in particular, about 95%, especially about 98% with the VKORC1 polypeptide according to one of SEQ ID No. 1, 12, 17, 21, 25, and 27, provided that such VKORC1 polypeptide has VKORC1 activity. Moreover, it is preferred that the term "VKORC1 polypeptide" also encompasses homologous polypeptides which originate from organisms other than human, preferably from non-human mammals such as, rodents, e.g. mouse, rats, or monkeys and pigs and other vertebrates and invertebrates, such as those amino acid sequences according to SEQ ID, Nos. 12, 17, 21, 25, 27, provided that such VKORC1 polypeptide has VKORC1 activity, It is even more preferred that the term "VKORC1 polypeptide" also includes VKORC1 polypeptides which are encoded by different alleles of the gene, in different individuals, in different organs of an organism or in different developmental phases, provided that such VKORC1 polypeptide has VKORC1 activity. It is further intended that the term "VKORC1 polypeptide" preferably also encompasses naturally occurring or synthetic mutations that exert no or only insignificant effects onto the activity of the VKORC1 polypeptide. Other polypeptides preferably encompassed by the term "VKORC1 polypeptide" include VKORC1 polypeptides that may arise from differential splicing of the VKORC1 transcript, provided that such VKORC1 polypeptide has VKORC1 activity.
  • The term "fragment of the polypeptide sequence" is intended to encompass partial sequences of VKORC1 polypeptides, which fragments comprise, preferably consist of at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95% of the full length sequence of the VKORC1 polypeptide. In particular, it is preferred that the fragment consists of a single contiguous sequence of the VKORC1 polypeptide but it may also contain at least two, at least three or at least about five different sequence portions of a VKORC1 polypeptide according to the invention which may or may not be interspaced by a heterologeous sequence or contain no extra polypeptide sequence at all.
  • The term "sequence homology" is understood as the degree of identity (% identity) of two sequences, that in the case of polypeptides can be determined by means of for example BlastP 2.0.1 and in the case of nucleic acids by means of for example BLASTN 2.014, wherein the Filter is set off and BLOSUM is 62 (Altschul et al., 1997).
  • "VKORC1 activity" within the meaning of the present invention is intended to mean the biological activity of the VKORC1 polypeptide of SEQ ID No.1. More preferably, "VKORC1 activity" is defined as the activity of the VKORC1 polypeptide to enzymatically convert (or support the enzymatic conversion) of vitamin K2,3-epoxide to vitamin K-quinone and/or the conversion of vitamin k quinone to vitamin K hydroquinone. VKORC1 activity may be determined using an assay based on the experiments described in detail in example 7 and figure 10. Using that assay, a measured percentage of vitamin K epoxide converted Into vitamin K quinone (product/substrate+product) in cells expressing a given VKORC1 polypeptide, or a nucleic acid molecule coding for such VKORC1 polypeptide, which raises the basal VKOR-actvity of HEK293 cells from about 1% (1.49 and 0.96% for untransfected and mock-transfected HEK293 cells, respectively) to about 15% or more, preferably to about 18% or more, preferably to about 20% or more, most preferably to about 25% or more is considered a VKORC1 activity within the meaning of the invention.
  • The VKORC1 polypeptides according to the present invention may be produced by a method described in more detail below. Among others, the VKORC1 polypeptides are useful for identifying coumarin derivatives that avoid the problems described above. In particular they are useful for identifying coumarin derivatives, that effectively inhibit VKORC1 activity and that In independent assays are tested for (1) their metabolic half life in order to identify coumarin derivatives that are metabolized faster than the coumarins known In the art, (2) their ability to cause skin necrosis to identify coumarin derivatives that do not cause skin necrosis or to a lesser extend than the coumarins known in the art, (3) coumarin derivative-drug interactions in order to identify coumarin derivatives with lesser side effects than the coumarins known in the art. Moreover, the VKORC1 polypeptides according to the present invention are useful for identifying a VKORC1 sequence interacting with coumarin and its derivatives, and for treating patients having a decreased or increased VKORC1 activity relative to control levels.
  • The term "VKORC1 nucleic acid" relates to RNA or DNA, which may be a single or preferably a double stranded molecule. The sequence of the VKORC1 nucleic acid may further comprise at least one intron and/or one polyA sequence. The term "VKORC1 nucleic acid" may also encompass a precursor stage, for example a propolypeptide or prepropolypeptide, thereof. It is also understood that untranslated sequences can be present at the 5' end and/or the 3' end of the nucleic acid, without the activity of the encoded polypeptides being significantly altered. However, the DNA region encoding the VKORC1 polypeptide is particularly preferred. In eukaryotes, this region begins with the first start codon (ATG) which is located in a Kozak sequence (Kozak, 1987) and extends to the next stop codon (TAG, TGA or TAA) which is located in the same reading frame as the ATG. in the case of prokaryotes, this region begins with the first AUG (or GUG) after a Shine-Dalgamo sequence and ends with the next stop codon (TAG, TGA or TAA) which is located in the same reading frame as the ATG. Moreover, the term "VKORC1 nucleic acid" may also encompass sequences which exhibit at least about 70%, in particular at least about 80%, especially at least about 90%, sequence homology with the sequence according to SEQ ID No. 2, 13, 18, 22, 26, and 28, preferably to the sequence according to SEQ ID No. 2, provided that the VKORC1 polypeptide encoded by such nucleic acid has VKORC1 activity. In a preferred embodiment of the invention the nucleic acid comprises a nucleic acid having a sequence complementary and/or antisense to a VKORC1 nucleic acid comprising, preferably consisting essentially of a nucleic acid sequence selected from the group consisting of:
    1. (a) a nucleic acid sequence coding for the VKORC1 polypeptide according to the invention:
    2. (b) a nucleic acid sequence selected from the group consisting of a sequence according to SEQ ID No. 2, 13, 18, 22, 26, and 28;
    3. (c) a nucleic acid sequence which hybridizes under stringent conditions to the nucleic acid sequence defined in (a) or (b), which nucleic acid sequence codes for a polypeptide having VKORC1 activity;
    4. (d) a nucleic acid sequence which, but for the degeneracy of the genetic code, would hybridize, preferably under stringent conditions, to the nucleic acid defined in (a), (b) or (c) and which nucleic acid sequence codes for a polypeptide having VKORC1 activity; and
    5. (e) a fragment of the nucleic add sequence defined in (a), (b), (c) or (d), which fragment codes for a polypeptides having VKORC1 activity.
  • Preferably, the VKORC1 nucleic acid encodes a target for coumarin and its derivatives in mammals. The VKORC1 nucleic acid may also comprises a non-functional mutant variant of the VKORC1 nucleic acid as defined above, such a variant containing a single nucleotide polymorphism (SNP) such as the nucleic acid sequences according to SEQ ID No. 8 and 9, provided that the VKORC1 polypeptide encoded by such nucleic acid has VKORC1 activity.
  • The term "stringent hybridization conditions" is to be understood, in particular, as meaning those conditions In which a hybridization takes place, for example, at 60°C in 2.5x SSC buffer followed by several washing steps at 37°C in a lower buffer concentration and remains stable.
  • The term "fragment of the nucleic acid sequence coding for a polypeptide having VKORC1 activity" is understood to encompass nucleic acid sequence fragments comprising, preferably consisting of at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95% of the full length sequence coding for VKORC1 polypeptide according to the invention, preferably coding for the polypeptide according to SEQ ID No. 1, provided that the polypeptide encoded by such fragment has VKORC1 activity. In particular, it is preferred that the fragment consists of a single contiguous sequence coding for the VKORC1 polypeptide but it may also contain at least two, at least three or at least about five different sequence portions, which may or may not be interspaced by a heterologeous sequence or contain no extra nucleic acid sequence at all, provided that all the sequence portions are arranged in the same reading frame. It is essential to the definition of these fragments that they display VKORC1 activity.
  • The VKORC1 nucleic acids can be produced by methods generally known to the skilled artisan. Nucleic acids may be prepared synthetically. Thus, the VKORC1 nucleic acids can, for example, be synthesized chemically, e.g. according to the phosphotriester method, with the aid of the DNA sequences as defined above and/or with the aid of the polypeptide sequences which are likewise defined above such as the SEQ ID No. 1 and by referring to the genetic code (see, e.g., Uhlmann, & Peyman, 1990). Preferably the VKORC1 nucleic acids are produced by recombinant gene technology methods generally known to the person skilled in the art.
  • Among others, the VKORC1 nucleic acids are useful (1) for identifying coumarin derivatives that avoid the problems described above, (2) for producing PCR primers, DNA and RNA probes, siRNA or shRNA, and for VKORC1 polypeptide, (3) for treating patients having a decreased or increased VKORC1 activity relative to control values, and (4) for identifying coumarin derivatives that may be employed for pest control of rodents, all of which are described In detail below.
  • As used herein, a host cell can be any host cell as defined below. Methods for selecting and culturing the host cells and for causing the host cells to express a polypeptide are generally known to the person skilled In the art. The same is true for methods of isolating the expressed polypeptide from the host cell; to this end an antibody according to the invention may be used for immunoaffinity precipitation. As an alternative the vector may contain a (poly)peptide tag that allows immunoaffinity precipitation by tag specific antibodies according to standard protocols known to the skilled worker (see also below).
  • It is also possible to add on at least one further "polypeptide tag" e.g. for the purpose of purifying the previously described VKORC1 polypeptides. For example, suitable protein tags enable the fusion proteins which are to be purified, to be absorbed with high affinity to a matrix. This is then followed, for example, by stringent washing with suitable buffers without eluting the fusion proteins to any significant extent, and, subsequently, specific elution of the fusion proteins. Examples of the protein tags which are known to the skilled person are a (His)6 tag, a Myc tag, a FLAG tag, a hemagglutinin tag, a glutathione transferase (GST) tag, intein having an affinity chitin-binding tag and a maltose-binding protein (MBP) tag. These protein tags can be located N-terminally, C-terminally and/or internally relative to the VKORC1 polypeptide. Fusion proteins are for example useful for the production of VKORC1 production and subsequent isolation. Moreover, the fusion proteins may be employed for detecting the localization of the expression product in the cell or the organism.
  • In order to enable the VKORC1 nucleic acids to be used according to the present invention they may be introduced into a eukaryotic or prokaryotic cell by means of transfection, transformation or infection, and thereby enable the polypeptide to be expressed. The VKORC1 nucleic acid can be present as a plasmid, or as a part of a viral or non-viral vector. Particularly suitable viral vectors in this connection are: baculoviruses, vaccinia viruses, adenoviruses, adeno-assoclated viruses and herpes viruses. Particularly suitable non-viral vectors are for example: virosomes, liposomes, cationic lipids and polylysine-conjugated DNA. The vectors can be prokaryotic or eukaryotic expression vectors. Examples of prokaryotic expression vectors are the pGEM vectors or pUC derivatives, which are used for expression in E. coli, and examples of eukaryotic expression vectors are the vectors p426Met25 or p426GAL1 (Mumberg et al., 1994) which are used for expression in Saccharamyces cerevisiae, the Baculovirus vectors, as disclosed in EP B1 0 127 839 or EP B1 0 549 721 , which are used for expression in insect cells, and the vectors Rc/CMV and Rc/RSV, or SV40 vectors, which are used for expression in mammalian cells, with all these vectors being generally available. In general, the expression vectors also contain promoters which are suitable for the respective cell, such as the trp promoter for expression in E. coli (see, e.g., EP-B1-0 154 133 ), the Met 25, GAL 1 or ADH2 promoter for expression in yeasts (Russel et al, 1983; Mumberg, see above), and the baculovirus polyhedrin promoter for expression in insect cells (see, e.g., EP B1 0127 839 ).
  • Promoters which permit constitutive, regulatable, tissue-specific, cell type-specific, cell cycle-specific or metabolism-specific expression in eukaryotic cells are suitable, for example, for expression In mammalian cells. Regulatable elements in accordance with the present invention are promoters, activator sequences, enhancers, silencers and/or repressor sequences. Examples of preferred regulatable elements which permit constitutive expression in eukaryotes are promoters which are recognized by RNA polymerase III or viral promoters, CMV enhancer, CMV promoter, SV40 promoter or LTR promoters, e.g. derived from MMTV (mouse mammary tumor virus; Lee et al., 1981) and other viral promoter and activator sequences which are derived from, for example, HBV, HCV, HSV, HPV, EBV, HTLV or HIV. Examples of regulatable elements which permit inducible expression in eukaryotes are the tetracycline operator in combination with an appropriate repressor (Gossen et al., 1994). The expression of VKORC1 nucleic acids preferably takes place under the control of tissue-specific promoters. The expression vectors may be used for preparing a VKORC1 polypeptides, DNA or RNA probes, or siRNA or shRNA, which can be used in accordance with the invention.
  • "Impaired VKORC1 activity" within the meaning of the invention relates to a level of activity and/or expression of the VKORC1 protein that is less than control level activity (as defined above) and/or expression determined in a healthy subject; the respective levels of activity may also be determined based on the assay as described in Example 7.
  • As used herein a host cell can be any cell suitable for expression of VKORC1 polypeptides and/or VKORC1 nucleic acids, preferably a HEK293-EBNA cell. Cells can be either prokaryotic or eukaryotic cells, heterologeous or autologous cells. Examples of prokaryotic cells are E. coll and examples of eukaryotic cells include primary hepatocytes cells, yeast cells, for example Saccharomyces cerevisiae or insect cells. More preferably the host cell is a cell line, e.g. a COS-cell such as COS-7 cells or hepatocytes cell lines such as HepG2 cells. Moreover the host cell Is preferably a non-human embryonic stem cell. Methods for selecting and culturing host cells and for causing the host cells to express a polypeptide are generally known to the person skilled in the art. Processes for the transformation of cells and/or stem cells are likewise well known to a person skilled in the art and include, for example, electroporation or microinjection. The host cells of the present invention can for example be employed for methods of identifying coumarin derivatives, for producing VKORC1 polypeptides and VKORC1 nucleic acids, siRNAs and shRNAs according to the invention, and for screening new drugs such as coumarin derivatives effecting VKORC1 activity and/or expression.
  • Transgenic animals in general show a tissue-specifically increased expression of the VKORC1 polypeptides and/or VKORC1 nucleic acids and can be used for the analysis of coagulation disorders and warfarin resistance and for development and evaluation of therapeutic strategies for such disorders. Transgenic animals may further be employed in the production of VKORC1 polypeptide. The polypeptide produced by the animal may for example be enriched in a body fluid of the animal.
  • Methods for the preparation of transgenic animals, in particular of transgenic mice, are likewise known to the person skilled in the art from DE 196 25 049 and US 4,736,866 ; US 5,625,122 ; US 5,698,765 ; US 5,583,278 and US 5,750,825 and include transgenic animals which can be produced, for example, by means of direct injection of expression vectors according to the present invention into embryos or spermatocytes or by injection of the expression vectors into the pronucleus of the fertilized ovum or by means of the transfection of expression vectors into embryonic stem cells or by nuclear transfer into appropriate recipient cells (Polites & Pinkert, 1994; Doetschman, in Pinkert, 1994, supra; Wood in Pinkert, 1994, supra; Monastersky in Pinkert, 1994, supra).
  • Within the meaning of the term "VKORC1 associated deficiency" Is intended to encompass a disorder or disease that is associated with warfarin resistance, i.e. the patient displays a reduced or abolished susceptibility to treatment with coumarin or its derivatives, preferably the warfarin resistance results from a sequence abnormality of the VKORC1 polypeptide. Moreover, the term preferably also encompasses disorders or diseases associated with a level of activity and/or expression of VKORC1 that differs significantly from the condition in healthy patients, preferably the expression of VKORC1 polypeptide and/or its activity is significantly reduced or abolished which can e.g. be determined by measuring the prothrombin time, e.g. by international normalized ration (INR) protocol. Such VKORC1 associated deficiency may be caused by a sequence abnormality in the VKORC1 polypeptide or VKORC1 nucleic acid as described in detail below. Moreover, when the level of expression and/or activity of VKORC1 polypeptide or VKORC1 nucleic acid is reduced or even completely abolished, gamma-carboxylation of vitamin K dependant proteins may also be impaired as well. Thus, in this context the term "VKORC1 associated deficiency" also encompasses diseases and/or disorder selected from familial multiple factor deficiency, a disorder or disease associated with decreased blood coagulation, such as hemophilia and a disorder associated decreased vascular calcification, diseases and/or disorders associated with impaired gamma-carboxylation of vitamin K dependant proteins.
  • It is also conceivable that the level of expression and/or activity of VKORC1 polypeptide will be increased relative to the condition in healthy patients. Such deficiencies which are also encompassed by the term the "VKORC1 associated deficiency" may be caused by a sequence abnormality in the VKORC1 polypeptide and/or the corresponding gene. Moreover, when the level of expression and/or activity of VKORC1 polypeptide or VKORC1 nucleic acid is increased, gamma-carboxylation of vitamin K dependant proteins may be facilitated as well. Thus, in this context the term "VKORC1 associated deficiency" may further comprise deficiencies selected from diseases or disorders associated with increased blood coagulation including patients suffering from a thrombus and/or patients having an increased risk of developing a thrombus, preferably due to a sequence abnormality in the VKORC1 polypeptide or its gene, diseases and/or disorders associated with improved gamma-carboxylation of vitamin K dependant proteins.
  • It is also possible that a sequence abnormality In the VKORC1 polypeptide and/or the corresponding gene may increase the susceptibility to treatment with coumarin and its derivatives in a patient having such sequence abnormality. As a result patients undergoing coumarin treating may show very low blood coagulation values. Such disorders associated with increased susceptibility to treatment with coumarin are also intended to be encompassed by the term "VKORC1 associated deficiency". Patients carrying a VKORC1 gene having a stop-mutation suffer from such deficiency associated with a increased coumarin sensitivity.
  • The term "sample" is intended to refer to a biomaterial comprising fetal or adult tissue or cell, preferably tissue or cells, preferably isolated or derived from heart, kidney and lung, pancreas, brain, placenta and skeletal muscle and blood, preferably from liver. The sample can be isolated from a patient or another subject by means of methods including invasive or non-invasive methods. Invasive methods are generally known to the skilled artisan and comprise for example isolation of the sample by means of puncturing, surgical removal of the sample from the opened body or by means of endoscopic instruments. Minimally invasive and non-invasive methods are also known to the person skilled in the art and include for example, collecting body fluids such as blood, preferably by venopuncture, or urine or feces. The term "sample" may also encompass a genomic or an expression library, preferably constructed based on an sample isolated from a patient, in which case techniques for isolation of the cDNA that are generally known to the skilled worker may be used.
  • According to the present invention, the term "antibody" or "antibody fragment" is understood as also meaning antibodies or antigen-binding parts thereof prepared by genetic engineering and optionally modified, such as, for example, chimeric antibodies, humanized antibodies, multifunctional antibodies, bi- or oligospecific antibodies, single-stranded antibodies, F(ab) or F(ab)2 fragments (see, for example, EP-B1-0 368 684 , US 4,816,567 , US 4,816,397 , WO 88/01649 . WO 93/06213 , WO 98/24884 ).
  • In another aspect the present invention provides a method of identifying a coumarin derivative which exerts an effect onto the activity of VKORC1 polypeptide as defined above, preferably a VKORC1 polypeptide comprising a polypeptide sequence selected from the group consisting of:
    1. (a) a polypeptide sequence selected from the group consisting of a sequence according to SEQ ID No. 1,12, and 17;
    2. (b) a polypeptide sequence of an allele of the polypeptide sequence defined in (a);
    3. (c) a polypeptide sequence having at least 80% homology with the polypeptide sequence defined in (a) or (b), wherein the polypeptide sequence has VKORC1 activity; and
    4. (d) a polypeptide sequence of a fragment of the polypeptide sequence defined in (a), (b) or (c) having VKORC1 activity,
    comprising the steps of:
    • (I) providing a host cell having been introduced the VKORC1 nucleic acid or a vector containing the VKORC1 nucleic acid;
    • (II) expressing the VKORC1 polypeptide in the host cell;
    • (III) administering a candidate coumarin derivative;
    • (IV) determining the activity of VKORC1 polypeptide (candidate activity value);
    • (V) comparing the candidate activity value with a control activity value; and
    • (VI) identifying the candidate coumarin derivative as a coumarin derivative exerting an effect onto the activity of the VKORC1 polypeptide, provided the candidate activity value is significantly different from the control activity value.
  • The the determined activity of VKORC1 polypeptide is dithiothreitol-dependent conversion of vitamin K 2,3-epoxide to vitamin K quinone and wherein the significantly different activity value is a candidate activity value which is significantly higher than the control activity value, as described in further detail above and in Example 7 and Figure 10. If essentially the same concentration of candidate coumarin derivative yields a lower percentage of vitamin K epoxide converted into vitamin K quinone (product/substrate+product) as warfarine does in this concentration, this is indicative ot the candidate coumarin derivative having a stronger inhibitory effect than warfarin, and vice versa.
  • Preferably in the method of identifying a coumarin derivative according to the present invention the control activity value is determined by a method comprising the steps of:
    1. (A) providing a host cell according to step (I);
    2. (B) expressing the VKORC1 polypeptide in the host cell; and
    3. (C) determining the activity of VKORC1 polypeptide (control activity value).
  • Even more preferably in the method of identifying a coumarin derivative according to the present invention at least one additional compound is introduced into the host cell, which compound is selected from the group consisting of vitamin K, cytochrome B5, a nucleic acid coding for gamma-glutamyl-carboxylase, for microsomal epoxidehydrolase, for calumenin, or for glutathion-S-transferase. Methods for introducing nucleic acids into host cells have been described in detail above. Preferably the nucleic acids are expressed under the control of a constitutively active promoter or a promoter which can be specifically activated in the host cell chosen.
  • The methods of identifying a coumarin derivative are useful for developing novel coumarin derivatives that avoid at least one of the limitations of coumarin and its derivatives known in the art. If analysis of the kinetics of blood coagulation is included as a separate assay into the determination of VKORC1 polypeptide activity, the method according to the invention may be useful in identifying coumarin derivatives which mediate blood coagulation faster than coumarin and its derivatives known in the art and/or that are metabolized more rapidly so that accumulation of coumarin and its derivatives may be prevented or ameliorated and as a result the danger of overdosing is substantially decreased or even abolished. Moreover, such method of identifying may be combined with other assays such that coumarin derivatives may be identified which have a stronger (weaker) effect onto VKORC1 activity and thus in turn onto the blood coagulation and which coumarin derivatives in independent assays prove (1) to be metabolized more rapidly so that accumulation of coumarin may be prevented or ameliorated and as a result the danger of overdosing is substantially decreased or even abolished, (2) not to cause or to cause to lesser extend skin necrosis in patients or embryopathy if applied during pregnancy, and/or (3) to be metabolized faster or to be less more stabil and/or to be affected less by other drugs like Phenobarbital or amiodarone. The assays which are suitable to screen for such properties of the coumarin derivatives and which are to be combined into a screen with the method of identifying a coumarin derivative according to the invention are generally known to the person skilled in the art.
  • The term "coumarin" is understood as meaning 3-(acetonylbenzyl)- 4-hydroxycoumarin, i.e. COUMADIN® or sodium warfarin.
  • The term "derivative of coumarin" is understood to encompass organic or inorganic compounds, peptides, polypeptides or complexes thereof, provided that they exert an effect onto the activity and/or expression of VKORC1 polypeptide, preferably an effect that inhibits the activity of the VKORC1 polypeptide, even more preferably a VKORC1 polypeptide-specific effect, i.e the coumarin derivative does not directly interact with other molecules involved in the coagulation pathway. Examples of such compounds are organic molecules that are derived from libraries of compounds, preferably those that have been analyzed for their pharmacological activity. On account of their interaction, the derivatives of coumarin can influence the activity of the VKORC1 polypeptide in vivo and/or in vitro and enter into interactions of covalent or non-covalent manner with them. If the coumarin derivative is a chiral compound it is understood that "derivative of coumarin" also encompasses the respective R- and L-enantiomeril forms of the compound like those disclosed in WO 00/43003 . In particular the term "derivative of coumarin" refers to compounds derived from 4-hydroxycoumarin, especially compounds derived from COUMADIN. More preferably, "derivative of coumarin" also includes any coagulants which inhibits the regeneration of active vitamin K.
  • The term "candidate coumarin derivative" is understood to encompass organic or inorganic compounds, peptides, polypeptides or complexes. Examples of such compounds are organic molecules that are derived from libraries of compounds, preferably those that have been analyzed for their pharmacological activity. Preferably the term refers to compounds that are structurally related or derived from 4-hydroxycoumarin, especially compounds related or derived from COUMADIN. If the candidate coumarin derivative is a chiral compound it is understood that the respective R- and L-enantiomeric forms of the compound like those disclosed in WO 00/43003 are also encompassed by the term "candidate coumarin derivative".
  • In another aspect the present invention provides a method of determining a VKORC1 polypeptide sequence which conveys a coumarin effect exerted onto VKORC1 activity, comprising the steps of:
    • (I) providing a cell expressing a VKORC1 polypeptide comprising a polypeptide sequence selected from the group consisting of:
      1. (a) a polypeptide sequence selected from the group consisting of a sequence according to SEQ ID No. 1, 12, and 17;
      2. (b) a polypeptide sequence of an allele of the polypeptide sequence defined in (a);
      3. (c) a polypeptide sequence having at least 80% homology with the polypeptide sequence defined in (a) or (b), wherein the polypeptide sequence has VKORC1 activity; and
      4. (d) a polypeptide sequence of a fragment of the polypeptide sequence defined in (a), (b) or (c) having VKORC1 activity,
      invention, preferably a polypeptide according to SEQ ID NO. 1, 12, 17, 21, which VKORC1 polypeptide has at least one sequence abnormality, preferably a sequence abnormality selected from the group consisting of V29L, V45A, R58G, R98W, L128R and Y139C;
    • (II) administering coumarin or a derivative thereof to the cell;
    • (III) determining the activity of the VKORC1 polypeptide (sequence abnormality activity value); and
    • (FV) comparing the sequence abnormality activity value with the control sequence activity value,
    wherein a significant deviation of the sequence abnormality activity value from the control sequence activity value is indicative that the sequence abnormality of the VKORC1 polypeptide conveys the coumarin effect exerted onto VKORC1 polypeptide. The activity of the VKORC1 polypeptide may be determined as described in detail above.
  • More preferably, in the method of determining a VKORC1 polypeptide sequence the control sequence activity value is determined by a method comprising the steps of:
    • (I) providing a cell expressing a VKORC1 polypeptide, comprising a polypeptide sequence selected from the group consisting of:
      1. (a) a polypeptide sequence selected from the group consisting of a sequence according to SEQ ID No. 1, 12, and 17;
      2. (b) a polypeptide sequence of an allele of the polypeptide sequence defined in (a);
      3. (c) a polypeptide sequence having at least 80% homology with the polypeptide sequence defined in (a) or (b), wherein the polypeptide sequence has VKORC1 activity; and
      4. (d) a polypeptide sequence of a fragment of the polypeptide sequence defined in (a), (b) or (c) having VKORC1 activity,
    • (II) administering coumarin or a derivative thereof to the cell;
    • (III) determining the activity of the VKORC1 polypeptide (control sequence activity value).
  • The determined VKORC1 activity is dithiothreitol-dependent conversion of vitamin K 2,3-epoxide to vitamin K quinone and the significantly different value is a sequence abnormality activity value which is significantly higher than the control sequence activity value. Further details are provided above and in Example 7 and Figure 10. The method may be useful in identifying VKORC1 polypeptides that are less sensitive to coumarins. By introducing VKORC1 polypeptides with different sequence abnormalities this method allows Identification of sites of the polypeptide which are critical for the interaction of a tested coumarin and VKORC1. Such knowledge will for example be useful for designing new coumarins.
  • it is particularly preferred in the method of determining a VKORC1 polypeptide sequence that at least one additional compound is introduced into the cell which compound is selected from the group consisting of vitamin K, cytochrome B5, and a nucleic acid coding for gamma-glutamyl-carboxylase, for microsomal epoxidehydrolase, for calumenin, or for glutathion-S-transferase.
  • A further aspect of the present invention relates to a VKORC1 polypeptide wherein the VKORC1 polypeptide contains at least one sequence abnormality, which exerts an effect on the activity of the VKORC1 polypeptide and wherein the VKORC1 polypeptide is the polypeptide according to SEQ ID No. 1 or 12 and the sequence abnormality is selected from the group consisting of V29L, V45A, R58G, R98W, L128R and Y139C.
  • Such VKORC1 polypeptide containing at least one sequence abnormality can be generated by methods generally known to the skilled worker, including recombinant techniques and e.g. site directed mutagenesis, or by isolation the VKORC1 polypeptide having the at least one sequence abnormality from a sample obtained from a patient, preferably from a patient suffering from VKORC1 associated deficiencies. Methods for isolating proteins from a sample have been described in detail above.
  • The VKORC1 polypeptide containing at least one sequence abnormality, which exerts an effect on the activity of the VKORC1 polypeptide can, for example, be used for generating antibodies binding specifically to these VKORC1 polypeptides. These antibodies in turn can be utilized for diagnosing VKORC1 associated deficiencies.
  • The term "sequence abnormality" is meant to encompass additions, insertions, deletions, substitutions of at least one amino acid that result in an alteration of the VKORC1 polypeptide sequence, preferably of the VKORC1 polypeptide sequence according to SE ID No. 1. Also encompassed are additions, insertions, deletions, substitutions of at least one nucleotide that lead to an altered amino acid sequence encoded by the VKORC1 nucleic acid sequence. Also encompassed are changes of the VKORC1 nucleic acid sequence that lead to a change in the reading frame of the nucleic acid sequence.
  • The sequence abnormalities are indicated in the single letter amino acid code with the original amino acid being placed to the left of the number indicating the number of the amino acid In the polypeptide sequence according to SEQ ID No. 1. The number to the right of the amino acid number indicates the amino acid that replaces the original amino acid. For example, the sequence abnormality V29L indicates that In position 29 of the VKORC1 polypeptide of SEQ ID No. 1 the amino acid valine (V) has been replaced by leucine (L). In case the sequence abnormality occurs in a VKORC1 polypeptide other than the VKORC1 polypeptide of SEQ ID No.1 the number in the code refers to the sequence position according to the numbering of the amino acids in that particular other polypeptide.
  • In another aspect the invention provides a method of diagnosing a warfarin resistance or multiple coagulation factor deficiency type 2 (VKCFD2) in a patient comprising the steps of:
    • (I) amplifying a DNA sample obtained from the patient or reverse transcribing a RNA sample obtained from the patient into a DNA and amplifying the DNA; and
    • (II) analyzing the amplified DNA of step (I) to determine at least one sequence abnormality in a nucleic acid sequence coding for a VKORC1 polypeptide or In an amino acid sequence of a VKORC1 polypeptide;
    wherein the determined sequence abnormality is indicative of the patient suffering from a VKORC1 associated deficiency; preferably Warfarine resistance; preferably the sequence abnormality exerts an effect on the activity of the VKORC1 polypeptide, preferably the sequence abnormality is selected from V29L, V45A, R58G, R98W, L128R, and Y139C.
  • Methods for obtaining samples from a patient and for isolating total RNA or mRNA are generally known to the skilled worker, some of which have been described above. Techniques for amplifying of DNA are not particularly limited and include PCR techniques which have also been described above. By the same token, techniques for reverse transcribing have been mentioned above and are not particularly limited and include reverse transcription using conventional protocols and commercially available kits that usually employ reverse transcriptase and oligo dT primers. The analysis may as well be based on genomic DNA isolated from a sample obtained from a patient.
  • Upon amplification, the DNA is subjected to analysis in order to determine at least one sequence abnormality in a nucleic acid sequence coding for the VKORC1 polypeptide. Methods for analyzing the amplified DNA are not particularly limited. Preferably the amplified DNA is analyzed by a technique selected from the group consisting of PCR-based analysis, preferentially using PCR primers specific for the sequence abnormality, restriction digestion analysis, and DNA sequencing analysis. In a preferred embodiment the nucleic acid carrying the sequence abnormality is coding for a VKORC1 sequence having a sequence abnormality is selected from the group consisting of V29L(85 G>T), V45A(134 T>C), R58G (172 A>G), R98W (292 C>T), and L128R (383 T>G), Y139C (416 A>G). In a preferred embodiment of the method of diagnosing the amplified DNA encodes at least a partial sequence of the VKORC1 polypeptide according to SEQ ID No. 1. One way of determining a sequence abnormality which is associated with a VKORC1 associated deficiency, is provided in Example 8.
  • The VKORC1 sequences according to the invention containing the mutations 85 G>T, 134 T>C, 172 A>G, 292 C>T, and 383 T>G, are provided in SEQ ID Nos. 3 to 7 and may be used as probes for diagnosing a VKORC1 associated deficiency using hybridization technique based analysis of nucleic acid samples obtained from a patient
  • As an alternative VKORC1 expression may be detected on the level of the VKORC1 polypeptide. Therefore, in another aspect the present invention provides a method of diagnosing a warfarin resistance or multiple coagulation factor deficiency type 2 (VKCFD2) in a patient comprising the steps of:
    • (I) providing a sample obtained from the patient; and
    • (II) detecting a VKORC1 polypeptide having a sequence abnormality in the sample using an antibody or a fragment thereof, which specifically recognizes and binds the VKORC1 polypeptide of claim 10
    wherein the determined sequence abnormality is indicative of the patient suffering from a VKORC1 associated deficiency. Preferably the sequence abnormality is selected from the group consisting of V29L, V45A, R58G, R98W, L128R, and Y139C.
  • Methods for obtaining samples have been described in detail already. Methods for detection of VKORC1 polypeptide having a sequence abnormality are not particularly limited, provided that the method allows specific detection of the protein carrying the sequence abnormality(ies). Examples of such methods preferably include immunohistochemical detection, immunoblotting, preferably Western blotting, and ELISA of the polypeptide, particularly preferred with antibodies specific for the VKORC1 polypeptide having a sequence abnormality as defined above. Analysis of sequence abnormalities on the level of the amino acid sequence is not particularly limited and may for example be carried out using VKORC1 antibodies which specifically recognize and bind a VKORC1 polypeptide having one or more sequence abnormalities.
  • Preferably the methods of diagnosing are used for diagnosing diseases and/or disorder selected from warfarin resistance, familial multiples factor deficiency, a disorder or disease associated with reduced or abolished blood coagulation, such as hemophilia and a disorder associated with decreased vascular calcification, diseases and/or disorders associated with impaired gamma-carboxylation of vitamin K dependant proteins.
  • Moreover, the methods of diagnosing may also be used to diagnose disorders and diseases associated with increased blood coagulation including patients and/or patients having an increased risk of developing a thrombus due to a sequence abnormality in the VKORC1 polypeptide or its gene, diseases and/or disorders associated with improved gamma-carboxylation of vitamin K dependant proteins.
  • in another aspect the present invention provides a method of identifying a coumarin derivative which exerts an inhibitory effect onto the activity of a VKORC1 polypeptide having at least one sequence abnormality, comprising the steps of:
    • (I) providing a cell expressing a VKORC1 polypeptide, wherein the VKORC1 polypeptide contains at least one sequence abnormality, which exerts an effect on the activity of the VKORC1 polypeptide, and wherein the VKORC1 polypeptide is the polypeptide according to SEQ ID No. 1 or 12 and the sequence abnormality is selected from the group consisting of V29L, V45A, R58G, R98W, L128R, and Y139C, preferably a polypeptide encoded by a sequence selected from the group consisting of 3, 4, 5, 6, 7, 14, and 94;
    • (II) administering a candidate coumarin derivative to the cell;
    • (III) determining the activity of the VKORC1 polypeptide (sequence abnormality activity value); and
    • (IV) comparing the sequence abnormality activity value with the control sequence activity value,
    • (V) identifying the candidate coumarin derivative as the coumarin derivative exerting an inhibitory effect onto the activity of a VKORC1 polypeptide, if the administration of the candidate coumarin derivative results in a sequence abnormality, activity value which is significantly lower than the control sequence activity value.
  • More preferably, the control sequence activity value is determined by a method comprising the steps of:
    • (I) providing a cell expressing the VKORC1 polypeptide of claim 1, preferably a polypeptide according to SEQ ID No. 1 or 12;
    • (II) administering coumarin to the cell;
    • (III) determining the activity of the VKORC1 polypeptide (control sequence activity value).
  • The activity of the VKORC1 polypeptide may be determined as described in detail above. And the method may be adopted from the method described in Example 7. Such method of identifying a coumarin derivative is useful for identifying coumarin derivatives that can be used as anticoagulants in warfarin resistance patients.
  • In another aspect the present invention provides a method of identifying a coumarin derivative which is toxicologically effective in warfarin-resistant rodents comprising the steps of:
    • (I) providing a warfarin-resistant rodent;
    • (II) administering a candidate coumarin derivative to the rodent;
    • (III) determining the toxicity of the candidate coumarin derivative onto the rodent (candidate coumarin derivative toxicity value);
    • (IV) comparing the candidate coumarin derivative toxicity value with a control coumarin toxicity value; and
    • (V) identifying the candidate coumarin derivative as a toxicologically effective coumarin derivative, provided that the candidate coumarin derivative toxicity value is significantly larger than the control coumarin toxicity value, wherein
    the warfarin-resistant rodent is a rodent transgenic for a VKORC1 polypeptide comprising a polypeptide sequence selected from the group consisting of:
    1. (a) a polypeptide sequence selected from the group consisting of a sequence according to SEQ ID No. 1, 12, and 17;
    2. (b) a polypeptide sequence of an allele of the polypeptide sequence defined in (a);
    3. (c) a polypeptide sequence having at least 80% homology with the polypeptide sequence defined in (a) or (b), wherein the polypeptide sequence has VKORC1 activity; and
    4. (d) a polypeptide sequence of a fragment of the polypeptide sequence defined in (a), (b) or (c) having VKORC1 activity,
    wherein the VKORC1 polypeptide contains at least one sequence abnormality, which causes warfarin resistance, preferably a polypeptide encoded by the sequence according to SEQ ID No. 14 or a commercially available Warfarine resistant rat or a wild catch rat having Warfarine resistance. More preferably, the VKORC1 polypeptide is the polypeptide according to SEQ ID No. 12 and the sequence abnormality is selected from the group consisting of V29L (85 G>T), V45A(134 T>C), R58G (172 A>G), R98W (292 C>T), and L128R (383 T>G), and Y139C (416 A>G).
  • The sample can be any organ, tissue, body fluid or probe provided that it contains genomic DNA or mRNA from the rat to be tested. Preferably the sample is blood, tissue from tail or ear urine or feces. Further details of the method are provided in Example 9.
  • Warfarin-resistant rodents have been described (Kohn & Pelz, 1999) and may be obtained from commercial suppliers (e.g. The Federal Biological Research Center for Agriculture and Forestry, Institute for Nematology and Vertebrate Research, Toppheideweg 88, 48161 Münster, Germany). In a preferred embodiment the warfarin-resistant rodent is a rodent transgenic for VKORC1 polypeptide containing at least one sequence abnormality, which sequence abnormality causes warfarin resistance. More preferred, the VKORC1 polypeptide containing at least one sequence abnormality is the polypeptide encoded by the nucleic acid sequence according to SEQ ID Nos. 3 to 7, and 15, and the sequence abnormality is selected from the group consisting of V29L (85 G>T), V45A(134 T>C), R58G (172 A>G), R98W (292 C>T), and L128R (383 T>G), Y139C (416 A>G). Methods for generating rodent, in particular mice transgenic for the recited VKORC1 polypeptides have been described above.
  • According to another aspect the invention relates to the use of PCR primers according to SEQ ID No. 88 to 91 for determining whether or not a rat has a warfarin resistance genotype in a sample obtained from a rat.
  • The administration of coumarin and its derivatives is not particularly limited. Typically coumarin (warfarin) -containing toxicologically effective compositions are formulated as granular bait compositions containing from about 50-300 ppm, preferably about 250 ppm, of coumarin and its derivatives. The bait is typically formulated with from 0.5% to 2.5% of warfarin concentrate in a suitable binder such as corn oil. If corn oil is used as a binder, it can be present in an amount of from about 0.5% to about 2% of the total composition. The binder and warfarin are then mixed in e.g. with a product based on cereal products, corn meal, rolled oats, mixed animal feeds, and similar products known in the art. The administration, i.e. the amount, formulation and frequency and duration of administration may follow standard protocols for assessing the toxicity of warfarin in rodents, more preferred follow standard protocols for assessing the lethal dose 50 (LD50) value for a given poison to be tested, all of which are generally known to the skilled person and described in example 9
  • In order for the coumarin derivative to be toxicologically effective it is desirable that multiple ingestions are required to kill the rodent so that they do not develop bait shyness. Therefore, it is preferred to repeat the administration of the candidate coumarin derivative compositions a number of times. Usually the rodents begin to die after four or five daily doses of the compositions. Moreover, it may be preferred to suppress pain in the rats in order to ameliorate the suffering during the experiments by administration of pain suppressing agents generally known to the skilled worker. Inclusion of pain-suppressors in the coumarin and coumarin derivative composition may further be advantageous in order to further suppress the chance that rodents develop bait shyness.
  • Following administration the toxicity of the candidate coumarin derivative onto the rodent is determined which yields the candidate coumarin derivative toxicity value. Methods for determining the toxicity of candidate coumarin derivatives are generally known to the skilled worker and include LD50 analysis, analysis of the blood coagulation by determining the prothrombin time, e.g. by international normalized ration (INR) protocol. The determined candidate coumarin derivative toxicity value is then compared with an appropriate control coumarin toxicity value determined on the basis of subjecting a different specimen of the warfarin-resistant rodent to the same treatment but exchanging the coumarin derivatives administration with an administration of a standard rodent coumarin compositions which the rodents are resistant for, commonly used for pest control. The same experimental conditions described above for the candidate coumarin derivative administration are used for the control. If the candidate coumarin derivative toxicity value is equal or preferably statistical significantly larger than the control coumarin toxicity value, the candidate coumarin derivative represents a toxicologically effective coumarin derivative.
  • The invention will now be further illustrated below with the aid of the figures and examples, representing preferred embodiments and features of the invention.
    • Examples Example 1: Characterization of the genomic candidate region
  • The locus for combined deficiency of vitamin K-dependent clotting factor type 2 (VKCFD2) to the pericentromeric region of chromosome 16 between the markers D16S3131 and D16S419 has been mapped [Fregin et al., 2002]. This region comprises approximately 20 Mb. The genes responsible for warfarin resistance in rats (Rw) and mice (War) had been mapped to chromosome 1 [Kohn et al., 1999] and chromosome 7[Wallace, 1976][Greavses & Ayres, 1967] in close linkage to the myosin light chain 2 gene (Myl2). The human ortholog of Myl2, HUMMLC2B, is located on chromosome 16p11 within the VKCFD2 candidate region and is part of a conserved linkage group of genes. Based on this synteny and on biochemical considerations, it is hypothesized that VKCFD2 and warfarin resistance may be due to allelic mutations in the same gene. If so, this would narrow down the critical interval in humans to a region of approximately 4.5 Mb between the interleukin 4 receptor gene (IL4R) and the integrin alpha M chain gene (ITGAM) on the short arm of chromosome 16 (Fig. 1).
  • According to the genome assembly, this region contains 141 Ensembl genes with approximately 1000 exons. Of these genes, 117 were annotated as known. Many of these genes could be excluded from further analysis because their function was well established and obviously not related to the metabolic steps of the vitamin K cycle. On the other side, genes upstream and downstream of this region are included that were regarded as functional candidates into the mutation screen.
    • Example 2: Mutation screening
  • Using genomic DNA from two VKCFD2 and three WR subjects, a systematic mutation screen was initiated by comparative sequencing of the remaining candidate genes. Clinical data of the VKCFD2 families have been described previously [Oldenburg et al., 2000]. Warfarin resistant patients were ascertained due to their abnormal response to oral warfarin administration during thrombosis treatment or prevention. Patient C and E are sporadic cases. Patient D has two brothers also suffering from warfarin resistance. Patients C and D required approximately 150 - 250 mg warfarin per week to achieve a therapeutic range of oral anticoagulation whereas patient E did not show any response at all. All patients gave informed consent before participating.
  • Surprisingly, missense mutations were found in a gene of unknown function in all investigated VKCFD2 and WR subjects (Fig. 2). This gene (IMAGE3455200) spans a genomic region of 5126 bp and comprises three exons coding for a protein of 163 amino acids. It was named vitamin K epoxide recycling protein 1 (VKORC1). Both non-related VKCFD2 patients and their affected siblings were found to harbor the same homozygous point mutation in the third exon (292C>T) whereas the parents were found to be heterozygous. The mutation is caused by the replacement of arginine by tryptophan at amino acid residue 98 (R98W). The families are of German and Lebanese origin. The haplotypes in the region of homozygosity around the mutated gene were different in both families indicating independent mutation events. In the WR patients, three different heterozygous mutations were found leading to a valine by leucine substitution (patient C: V29L), an arginine by glycine substitution (patient D: R58G) and an exchange of leucine to arginine (patient E: L128R). The R58G mutation is shared by the two affected brothers of index patient D. The missense mutations were not present in 384 control chromosomes. Sequencing of the control chromosomes revealed two non-synonymous single nucleotide polymorphisms (C43C; L120L).
  • Genome sequences and annotation were obtained from NCBI, UCSC and Ensembl. Primers for mutation screening were designed using Primer3 software integrated into a script, ExonPrimer, to allow automatic primer design. For mutation screening, exons with intronic primers were amplified and amplified fragments were analyzed by direct sequencing with the BigDye Terminator Cycle sequencing kit (ABI)). Primer sequences were available on request. Topology predictions were performed using TMPRED and TMHM.
    • Example 3: Homology and protein structure
  • An orthologue of the VKORC1 gene was present in mouse (NM_178600) and the orthologues in rat and in Fugu rubripes were established by homology searches and RT-PCR (Fig. 3). The corresponding proteins share 79% to 84% identity with the human protein. Database searches did not show any homology to a known gene nor to any characterized protein domain. Topology prediction programs anticipated three transmembrane domains (TM). The first TM is placed between residues 10 to 29 by all programs tested. The predictions are discordant for the second and the third TM, which are located between amino acids 100 and 150.. The PSORT II server predicted an ER membrane retention signal (KKXX or KXKXX) at position 159-163 of humans VKORC1 with a probability of 67 %[Jackson et al., 1990]. The consensus sequence was also present in the other VKORC1 proteins. This is in accordance with the likely location of the VKORC1 complex within the ER membrane system[Cain et al., 1997].
  • Tblastn searches with VKORC1 detected a homologous human (BC027734) and mouse gene (AK009497) showing 50% protein identity each. Both mRNA were wrongly predicted to code for proteins that show no homology with VKORC1. The predicted human protein starts at the third methionine. The mouse mRNA sequence is incomplete with a protein predicted in a different reading frame. The complete cDNA was established in mouse as well as in Fugu rubripes and partially in rat. These proteins were designated VKORC1 like protein 1 (VKORC1L1). Human, mouse and rat VKORC1L1 proteins share, approximately 84% identity between each other and approximately 50% identity with the corresponding VKORC1 proteins. A homologous protein was further detected in Xenopus laevis (AAH43742) and - with weaker homology (1 e-14) - in Anopheles gambiae (EAA06271). Tree analysis suggested that both these proteins are orthologues to the VKORC1 gene.
    • Example 4: Expression analysis
  • VKORC1 seems to be widely expressed. The corresponding Unigene entry contains more than 100 ESTs in various tissues. The expression of VKORC1 in fetal and adult human tissues is examined by Northern blot analysis. To this end Human multiple tissue northern blots (Fetal Blot 1, Stratagene; Human 12-Lane, BD Clontech) contained 2 µg of poly(A)+-RNA. Full-length human VKORC1 cDNA was radiolabeled using random primers DNA labeling system (Invitrogen life technologies) and hybridized using miracleHyb High-Performance Hybridization Solution (Stratagene). A β-actin probe supplied with the multiple tissue northern blot was used for control hybridization.
  • The highest VKORC1 expression levels can be observed in fetal and adult liver (Fig. 4). High expression levels were also observed in fetal heart, kidney and lung, as well as in adult heart and pancreas. Fetal brain, adult placenta and skeletal muscle showed intermediate levels of expression. Minor expression levels were detected in adult brain, lung and kidney.
    • Example 5: Cloning of VKORC1 and construction of expression vectors
  • Amplification of the complete coding sequence of VKORC1 was performed from human liver and kidney cDNA (Marathon-Ready cDNA, BD Biosciences Clontech) with the following primers including cleavage sites for HindIII and EcoRI: VKORC1-HindIII-F: ATTAAGCTTCACCATGGGCAGCACCTGGGGGAGCCCT (SEQ ID No. 53)
    VKORC1-EcoRI-R: ATTGAATTCCGTGCCTCTTAGCCTTGCCCTG (SEQ ID No. 54).
    The product was cloned into the pBluescript II vector (Stratagene) that was cleaved with the corresponding restriction enzymes and verified by direct sequencing. For immunocytochemistry experiments, the insert was re-cloned into the mammalian expression vectors pEGFP-N1 (BD Biosciences Clontech) and pcDNA3.1/myc-His (Invitrogen).
  • For expression studies, the VKORC1 cDNA was cloned into the pcDNA3 vector (Invitrogen) after amplification with the primers VKORC1-pcdna3-F: GGGCGGAAGCTTGAGATAATGGGCA (SEQ ID No. 92) and VKORC1-pcdna3-R: GCTTGAATTCAGGGCTCAGTGC (SEQ ID No. 93). Mutagenesis was performed using the QuikChange mutagenesis Kit (Stratagene). Wildtype and mutated cDNAs were re-cloned for expression in pCEP4 (Invitrogen) using the HindIII and XhoI-sites. All constructs were verified by sequencing.
    • Example 6: Cell culture, transient transfection and immunocytochemistry and subcellular localization
  • From biochemical fractionation experiments it is known that the VKORC1 activity purifies with the microsomal membrane fraction [Cain et al., 1997]. Furthermore, the gamma-glutamyl-carboxylase has been localized to the membrane of the endoplasmic reticulum by immunocytochemistry [Presnell, 2002 #31]. In order to study the subcellular localization of human VKORC1 GFP- and myc-epitope tagged VKORC1 fusion protein constructs were generated for transient transfection experiments of COS-7 cells. Primary antibodies against the epitope tags and fluorochrome labeled secondary antibodies were used to visualize the fusion proteins. An antibody against the ER-specific protein calnexin served as a control. To this end COS-7 cells (DSMZ, Braunschweig) were maintained in Dulbecco's modified eagle's medium with 10% fetal calf serum. Cells were plated on glass cover slips in six-well plates and after 18-24 h in culture transfected with the expression vector constructs using Effectene (QIAGEN) according to the manufacturer's specifications. After 48-60 h of further culturing, the cells were washed with PBS and fixed in 70% acetone / 30% methanol at -20 °C for 15 min. Following fixation, the cells were permeabilized in PBS, 0.1% Nonidet P-40 (SIGMA N-6507), and then blocked with PBS, 2% BSA and 0.1% NP-40 at 37°C. Primary antibodies, Living Colors A.v. (JI-8) (BD Biosciences Clontech), anti-myc antibody (Invitrogen), and anti-calnexin (SIGMA) were diluted (1:100) in the blocking solution and incubated for 45 min at 37 °C. Cover slips were washed in PBS, 0.1% NP-40 for 30 min. The same incubation and washing procedures were used for the secondary antibodies, i.e. anti-mouse-IgG-FITC (SIGMA) and anti-rabbit-IgG F(ab')2 fragment-Cy3 (SIGMA). Cover slips were counterstained with DAPI (1:500) for 1 min, washed with deionized water, mounted on slides using Vectashield (Vector) and visualized using a Leica fluorescence microscope.
  • The green immunofluorescence of the VKORC1 fusion proteins decorated the mesh-like structures of the ER within the cytoplasm and perfectly co-localized with the label of the ER-marker calnexin (red) (Fig. 5).
    • Example 7: An assay for determining enzymatic VKORC1 activity
  • HEK293-EBNA cells (Invitrogen) were grown in MEM with 10% FCS. For each experiment, 6x105 cells were plated onto 94 mm Petri dishes. After 30 h at 37°C and 5% CO2, transfection (20 µg of DNA construct per dish) was performed using the calcium phosphate method. After 40 h at 35 °C, 3% CO2 transfected cells (nearly grown to confluence) were washed in PBS, harvested and lysed in 450µl 0.25 mM imidazole, (pH = 7.6), 0.5% CHAPS. Transfection efficiency was checked by sequencing of RT-PCR products of an aliquot of cells.
  • VKOR enzymatic activity was measured with 30 µl of the whole-cell extracts which were resuspended in 500 µl buffer A (0.25 mM imidazole, (pH = 7.6), 0.5% CHAPS). Then 20µl 125mM DTT were added with one minute incubation. Then 5µl 400 mM CaCl2, and Warfarin in 10µl DMSO (final concentration 0-80µM) were added. The reaction was started by addition of 2µl vitamin K2,3-epoxide (final concentration 5µM) and incubated at 30°C for one hour. Reaction was stopped by extraction of the substrate (Vitamin K2,3-epoxide) and the reaction products (Vitamin K-quinone and hydroquinone) using 1 ml 2-Propanol/Hexane (3:2, v/v); the organic supernatant was collected, dried and resolved in 50µl methanol and analyzed with an HPLC at 254 nm. The vitamin K quinone was separated from the epoxide by HPLC on a reversed phase C-18 column. During the extraction procedure vitamin K hydroquinone was quantitatively oxidized to the quinone form. The output of the HPLC was analyzed automatically by calculating the area under the line of extinction of each peak. The percentage of conversion of substrate was estimated by setting the area of the residual substrate-peak (epoxide) plus the product-peak (quinone) as 100 percent. Measurements were run in duplicate and the activity is given as percent of substrate converted into quinone. Vitamin K 2,3-epoxide was prepared by oxidation of vitamin K quinone (Sigma-Aldrich) with H2O2. Warfarin (Sigma-Aldrich) was added in DMSO (< 1 Vol%).
  • Dose-response to warfarin inhibition was measured at 5 to 80 µM final concentration (Walin & Martin 1985). Untransfected and mock-transfected cells showed a low basal activity which was warfarin sensitive. Overexpression of Wildtype VKORC1 resulted in a striking stimulation of VKOR activity. Production of vitamin K quinone was 14 to 21-fold increased compared to untreated and mock-transfected cells. The activity was inhibited by warfarin in a dose-dependent manner (Figure 10).
  • We also determined VKOR activity after transfection with mutated VKORC1 constructs (Figure 10). Recombinant expression of the R98W mutation observed in the two VKCFD2 families did only slightly increase VKOR activity in HEK293 cells. Spontaneous bleeding episodes and high serum levels of vitamin K epoxide in these patients suggest that the efficiency of vitamin K recycling is also drastically decreased in vivo (Oldenburg et al. 2000). The five WR mutations showed a reduced VKOR activity ranging from 5% in the L128R variant to 96% in the V29L mutation. Mutations V45A, R58G and Y139C displayed about 23%, 21 % and 48% activity, respectively (Table 1). Reduced VKOR activity associated with higher vitamin K demand and death from spontaneous bleeding has been observed in heterozygous and homozygous Rw rats (Martin et al. 1979, Thijssen & Pelz 2001, Fasco et al. 1983b). Similarly, in our expression system which mimics homozygous conditions WR mutations led to a lower functional efficiency of the VKOR complex. Whereas at the phenotypic level all WR variants exhibited at least partial resistance towards the anticoagulation effect of warfarin, both Wildtype and mutant proteins were sensitive to warfarin in vitro. At concentrations above 20 µM, mutations V29L and Y139C retained higher VKOR activities than the Wildtype while in mutations V45A, R58G, L128R, VKOR activity fell below the detection limit (Figure 10).
    • Example 8: A method of diagnosing a VKORC1 sequence abnormality
  • Genomic DNA of the specimen (human patient or mammal) is isolated according to standard procedures generally known to the skilled worker. The genomic DNA of the desired Exon (1-3) of VKORC1 is amplified by PCR using specific primers which can also be designed by the skilled artisan. The PCR-product is then purified using e.g. SAP/Exo (shrimp alkaline phosphatase and exonuclease) under standard conditions.
    The purified DNA is then subjected to standard sequencing procedures such as: addition of 0.3µl primer which is 10 pmol/µl (forward or reverse primer) to 1µl of the purified PCR-product; followed by addition of 8µl DTCS-Mix (Beckman-Coulter) and 10.7µl water; followed by cycle sequencing at
    First delay 96°C 60 sec
    Danaturation
    95°C 30 sec
    Annealing Primerspecific(55-60°C) 30 sec
    Elongation
    60°C 4 min
  • After the cycle sequencing purification by precipitation follows:
    • ad 2µl 100mM EDTA, 2µl 5M NaOAc (pH 4.8),1 µl Glycogen vortex
    • ad von 60 µl 95 % ethanol, vortex
    • centrifugation at 13000 g (10 min)
    • remove supernatant
    • wash pellet with 180 µl 70 % Ethanol
    • dry pellet
    • resolve pellet in 35 µl Sample Loading Solution (SLS)
  • Then the probes are pipetted on a microtiter plate overlaid with a drop of paraffin-oil. Then separation in the sequencer at 4,2 V for 60-120 min follows. The raw data is analyzed and the sequences are aligned with control sequences using the CEQ 2000 XL software (Version 4.3.9, Beckman Coulter). Differences between the control sequences (preferably the genomic VKORC1 nucleic acid sequence or its coding sequence according to SEQ ID NO. 2) and the sequenced DNA is indicative of the probes sequence to represent a VKORC1 nucleic acid containing a sequence abnormality.
    • Example 9: PCR-based assay for determining warfarin resistance in rats
  • In order to determine whether or not a rat (Rattus norvegicus) is warfarin resistant, i.e. whether the VKORC1 coding sequence according to SEQ ID No. 13 carries a mutation Y139C (416A>G), the following assay based on ARMS-PCR was employed using rat feces as a source of rat genomic DNA.
  • It is the principle of the assay to include into the PCR reaction (1) one PCR primer (rVKORC1-innerF) that specifically hybridizes to the DNA sequence which contains the warfarin resistant mutated allele 416G and (2) another PCR primer (rVKORC1-innerR) which specifically hybridizes to the Wildtype DNA sequence which contains the Wildtype allele 416A. Moreover, these two primers are oriented in opposite direction such that they pair with one out of two additional PCR primers included into the reaction. The latter primers are located in different distances to and in opposite direction relative to the 416 site and as a result, depending on whether the 416 site is mutated or not either the rVKORC1-innerR primer or the rVKORC1-innerF primer will anneal and the PCR reaction will result in amplified DNA of a different size which is indicative of the genotype of the rat which DNA has been analyzed. In Wildtype rats the PCR reaction will result in a band of 123 bp, whereas in rats homozygous to the mutation 416G the PCR reaction will yield a band at 101 bp. Finally in rats with a heterozygous genotype, the PCR reaction will give rise to two bands, one at 101 and another band at 123 bp.
  • The genomic DNA was isolated from the feces using standard DNA isolation procedures generally known to the skilled artisan. The following components were combined to a PCR reaction: 1 µl DNA (rat), 1 µl 5M Betain (Sigma), 2 pmol outer-Primer-F (1 µl of a 1:50-dilution), 2 pmol outer-Primer-R (1 µl of a 1:50-dilution), 10 pmol inner-Primer-F (1 µl of a 1:10-dilution), 10 pmol inner-Primer-R (1 µl of a 1:10- dilution), 0,25 µl Taq/Pfu-Polyme rase (1,25U Taq (Invitrogen) and 0,25 U Pfu (Stratagene)), ad 25 µl PCR-buffer (1 ml PCR-buffer contains: 100 µl 10xPCR-buffer (Invitrogen), 160 µl nucleotide stem-solution (1,25 mM dNTPs), 30 µl MgCl2, 610 µl aqua dest). The PCR conditions were: 95°C for 3 min, followed by 32 cycles of: 95°C for 20 sec, 62°C for 20 sec, and 70°C for 10 sec. Finally, the reaction is incubated at 70°C for 3 min. The PCR Products were separated by gel electrophoresis on a 3,5 % TAE-Agarose-Gel with ethidium bromide (10 µl of a 1 %-stem solution for every 100ml). The gelelectrophosesis was allowed to run for 30 min at 130 V.
  • The following primers were used:
    • rVKORC1-outerF: ATC CTG AGT TCC CTG GTG TCT GTC GCT G (SEQ ID No. 88)
    • rVKORC1-outerR: TCA GGG CTT TTT GAC CTT GTG TTC TGG C (SEQ ID No. 89)
    • 416G-mutant allele-specific PCR primer "rVKORC1-innerF": TGA TTT CTG CAT TGT TTG CAT CAC CAC ATG (SEQ ID No. 90)
    • 416A-wildtype allele-specific PCR primer "rVKORC1-innerR": CAA CAT CAG GCC CGC ATT GAT GGA AT (SEQ ID No. 91)
  • Rats (Rattus norvegicus) with and without warfarin resistance were used for the assay. The results of the PCR are shown in Figure 13. Wildtype rats exhibited a band at 123 bp, rats homozygous to the mutation exhibited a band at 101 bp and finally, rats with the heterozygous mutation showed two bands, one at 101 and another band at 123 bp.
  • As a result, this data demonstrate, that this assay can be employed to determine whether a given rat is warfarin resistant or not. Such assays are highly versatile in order to manage pest control in a given region, since knowledge of the frequency of warfarin resistant rats is critical for deciding which pesticide may be employed effectively. If in a given region there is a high prevalence of warfarin resistant rats warfarin and analogues thereof are an unsuitable means to kill the rats. If, however, the determined frequency of warfarin resistant rats is low, warfarin may be effectively used to fight rodents.
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    SEQUENCE LISTING
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      Oldenburg, Johannes
      Müller-Reible, Clemens
      Fregin, Andreas
      Rost, Simone
      Strom, Tim
    • <120> Vitamin K epoxid recycling polypeptide VKORC1, a therapeutic target of coumarin and their derivatives
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    • <400> 33
      gucucucgcu gguucugucu u    21
    • <210> 34
      <211> 21
      <212> RNA
      <213> Artificial sequence
    • <220>
      <223> siRNA sequence 4 homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1)
    • <400> 34
      uucagagagc gaccaagaca g    21
    • <210> 35
      <211> 57
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> Downstream primer 3 for homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1) siRNA
      sequence
    • <400> 35
      caaaaactgt aaaaagtctc tcgctggttc tgtcggtgtt tcgtcctttc cacaaga    57
    • <210> 36
      <211> 57
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> Downstream primer 4 for homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1) siRNA
      sequence
    • <400> 36
      caaaaactgt aaaaagacag aaccagcgag agacggtgtt tcgtcctttc cacaaga    57
    • <210> 37
      <211> 21
      <212> RNA
      <213> Artificial sequence
    • <220>
      <223> siRNA sequence 5 homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1)
    • <400> 37
      ggccuggauc cuguucuucu u    21
    • <210> 38
      <211> 21
      <212> RNA
      <213> Artificial sequence
    • <220>
      <223> siRNA sequence 6 homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1)
    • <400> 38
      uuccggaccu aggacaagaa g    21
    • <210> 39
      <211> 57
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> Downstream primer 5 for homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1) siRNA
      sequence
    • <400> 39
      caaaaactgt aaaaaggcct ggatcctgtt cttcggtgtt tcgtcctttc cacaaga    57
    • <210> 40
      <211> 57
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> Downstream primer 6 for homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1) siRNA sequence
    • <400> 40
      caaaaactgt aaaaagaaga acaggatcca ggccggtgtt tcgtcctttc cacaaga    57
    • <210> 41
      <211> 21
      <212> RNA
      <213> Artificial sequence
    • <220>
      <223> siRNA sequence 7 homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1)
    • <400> 41
      gauccuguuc uucgugcucu u    21
    • <210> 42
      <211> 21
      <212> RNA
      <213> Artificial sequence
    • <220>
      <223> siRNA sequence 8 homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1)
    • <400> 42
      uucuaggaca agaagcacga g    21
    • <210> 43
      <211> 57
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> Downstream primer 7 for homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1) siRNA
      sequence
    • <400> 43
      caaaaactgt aaaaagatcc tgttcttcgt gctcggtgtt tcgtcctttc cacaaga    57
    • <210> 44
      <211> 57
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> Downstream primer 8 for homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1) siRNA
      sequence
    • <400> 44
      caaaaactgt aaaaagagca cgaagaacag gatcggtgtt tcgtcctttc cacaaga    57
    • <210> 45
      <211> 21
      <212> RNA
      <213> Artificial sequence
    • <220>
      <223> siRNA sequence 9 homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1)
    • <400> 45
      gcauuguuug uaucaccacu u    21
    • <210> 46
      <211> 21
      <212> RNA
      <213> Artificial sequence
    • <220>
      <223> siRNA sequence 10 homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1)
    • <400> 46
      uucguaacaa acauaguggu g    21
    • <210> 47
      <211> 57
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> Downstream primer 9 for homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1) siRNA
      sequence
    • <400> 47
      caaaaactgt aaaaagcatt gtttgtatca ccacggtgtt tcgtcctttc cacaaga    57
    • <210> 48
      <211> 57
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> Downstream primer 10 for homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1) siRNA
      sequence
    • <400> 48
      caaaaactgt aaaaagtggt gatacaaaca atgcggtgtt tcgtcctttc cacaaga    57
    • <210> 49
      <211> 21
      <212> RNA
      <213> Artificial sequence
    • <220>
      <223> siRNA sequence 11 homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1)
    • <400> 49
      gcucaguuuc cggaaggucu u    21
    • <210> 50
      <211> 21
      <212> RNA
      <213> Artificial sequence
    • <220>
      <223> siRNA sequence 12 homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1)
    • <400> 50
      uucgagucaa aggccuucca g    21
    • <210> 51
      <211> 57
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> Downstream primer 11 for homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1) siRNA
      sequence
    • <400> 51
      caaaaactgt aaaaagctca gtttccggaa ggtcggtgtt tcgtcctttc cacaaga    57
    • <210> 52
      <211> 57
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> Downstream primer 12 for homo sapiens vitamin K epoxide reductase complex subunit 1 (Hs_VKORC1) siRNA
      sequence
    • <400> 52
      caaaaactgt aaaaagacct tccggaaact gagcggtgtt tcgtcctttc cacaaga    57
    • <210> 53
      <211> 37
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for amplification of the complete coding sequence of homo sapiens vitamin K epoxide reductase complex subunit 1 (VKORC1-HindIII-F)
    • <400> 53
      attaagcttc accatgggca gcacctgggg gagccct    37
    • <210> 54
      <211> 31
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for amplification of the complete coding sequence of homo sapiens vitamin K epoxide reductase complex subunit 1 (VKORC1-EcoRI-R)
    • <400> 54
      attgaattcc gtgcctctta gccttgccct g    31
    • <210> 55
      <211> 18
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Hs_VKORC1-Ex1-F
    • <400> 55
      caatcgccga gtcagagg    18
    • <210> 56
      <211> 20
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Hs_VKORC1-Ex1-R
    • <400> 56
      taatcatctg gcatcctggc    20
    • <210> 57
      <211> 19
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Hs_VKORC1-Ex2-F
    • <400> 57
      caaggcactg ggttgacag    19
    • <210> 58
      <211> 18
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Hs_VKORC1-Ex2-R
    • <400> 58
      gagtggggct gagctgac    18
    • <210> 59
      <211> 20
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Hs_VKORC1-Ex3-F
    • <400> 59
      gacatcatgg agtgttcggg    20
    • <210> 60
      <211> 20
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Hs_VKORC1-Ex3-R
    • <400> 60
      cttaggcaag gctcacatgc    20
    • <210> 61
      <211> 18
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Hs_VKORC1-cDNA-F
    • <400> 61
      ggcacgaggg ttttctcc    18
    • <210> 62
      <211> 19
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Hs_VKORC1-cDNA-R
    • <400> 62
      ctcacatgcc aaagcaaag    19
    • <210> 63
      <211> 18
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Rn_VKORC1-cDNA-F
    • <400> 63
      ggcgggttct tccctctt    18
    • <210> 64
      <211> 20
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Rn_VKORC1-cDNA-R
    • <400> 64
      catgtgctaa ggcaaagcaa    20
    • <210> 65
      <211> 21
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Rn_VKORC1-cDNA-F-nes
    • <400> 65
      ttgtgtctgc gctgtactgt c    21
    • <210> 66
      <211> 18
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Rn_VKORC1-cDNA-R-nes
    • <400> 66
      gtcagcctgg catgaggt    18
    • <210> 67
      <211> 22
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Fr_VKORC1-cDNA-F
    • <400> 67
      tcttttccat ttgattggtc ct    22
    • <210> 68
      <211> 21
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Fr_VKORC1-cDNA-R
    • <400> 68
      tcagtttagt cgcacctcct g    21
    • <210> 69
      <211> 20
      <212> DNA
      <213> Artificial,sequence
    • <220>
      <223> PCR primer for Fr_VKORC1-cDNA-F-nes
    • <400> 69
      gtggccatct gagcagaaac    20
    • <210> 70
      <211> 20
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Fr_VKORC1-cDNA-R-nes
    • <400> 70
      tgctggattt cagtgggaac    20
    • <210> 71
      <211> 18
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Hs_VKORC1L1-cDNA-F
    • <400> 71
      tgggtcgggc cccgacgg    18
    • <210> 72
      <211> 22
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Hs_VKORC1L1-cDNA-R
    • <400> 72
      tttaaatcca tcggctaaaa ac    22
    • <210> 73
      <211> 16
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Hs_VKORC1L1-cDNA-F-nes
    • <400> 73
      ggcggctgag gtggag    16
    • <210> 74
      <211> 21
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Hs_VKORC1L1-cDNA-R-nes
    • <400> 74
      agcaatggtt gctcacttta c    21
    • <210> 75
      <211> 20
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Fr_VKORC1L1-CDNA-F
    • <400> 75
      cgagctccct gcgtatgtat    20
    • <210> 76
      <211> 26
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Fr_VKORC1L1-cDNA-R
    • <400> 76
      gacgttgttg tttgtttatt tgattt    26
    • <210> 77
      <211> 21
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Fr_VKORC1L1-cDNA-F-nes
    • <400> 77
      cgtatgtatg cgtgtctcca g    21
    • <210> 78
      <211> 18
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Fr_VKORC1L1-cDNA-Rnes
    • <400> 78
      ttttcaccgc cgttctga    18
    • <210> 79
      <211> 18
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for PCR primer for Rn_VKORC1L1-cDNA-F
    • <400> 79
      ggcggcgtct gagtggag    18
    • <210> 80
      <211> 20
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Rn_VKORC1L1-cDNA-R
    • <400> 80
      acaggtttaa atccatcggc    20
    • <210> 81
      <211> 17
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Rn_VKORC1L1-cDNA-F-nes
    • <400> 81
      ctgagtggag gcggagg    17
    • <210> 82
      <211> 24
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Rn_VKORC1L1-cDNA-R-nes
    • <400> 82
      tttcatgttc atgatcacat tttg    24
    • <210> 83
      <211> 20
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Rn_VKORC1L1-cDNA-R-short
    • <400> 83
      ctggctgtca tgccaagtaa    20
    • <210> 84
      <211> 18
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Mm_VKORC1L1-cDNA-F
    • <400> 84
      ggaagatggc ggcgcccg    18
    • <210> 85
      <211> 20
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Mm_VKORC1L1-cDNA-R
    • <400> 85
      tctgctgtca acactgcacc    20
    • <210> 86
      <211> 17
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Mm_VKORC1L1-cDNA-F-nes
    • <400> 86
      ggtatgcagt gtgcgcc    17
    • <210> 87
      <211> 20
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> PCR primer for Mm_VKORC1L1-cDNA-R-nes
    • <400> 87
      gcatttccca agatgttctg    20
    • <210> 88
      <211> 28
      <212> DNA
      <213> Artificial Sequence
    • <220>
      <223> rVKORC1-outerF PCR-primer
    • <400> 88
      atcctgagtt ccctggtgtc tgtcgctg    28
    • <210> 89
      <211> 28
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> rVKORCI-outerR PCR-primer
    • <400> 89
      tcagggcttt ttgaccttgt gttctggc    28
    • <210> 90
      <211> 30
      <212> DNA
      <213> Artificial Sequence
    • <220>
      <223> rVKORC1-innerF PCR-primer
    • <400> 90
      tgatttctgc attgtttgca tcaccacatg    30
    • <210> 91
      <211> 26
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> rVKORC1-innerR PCR-primer
    • <400> 91
      caacatcagg cccgcattga tggaat    26
    • <210> 92
      <211> 25
      <212> DNA
      <213> artificial sequence
    • <220>
      <223> VKORC1-pcdna3-F PCR-primer
    • <400> 92
      gggcggaagc ttgagataat gggca    25
    • <210> 93
      <211> 22
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> VKORC1-pcdna3-R PCR-primer
    • <400> 93
      gcttgaattc agggctcagt gc    22
    • <210> 94
      <211> 492
      <212> DNA
      <213> Artificial sequence
    • <220>
      <223> Homo sapiens VKORC1 with rat warfarin resistance mutation 416A>G
    • <400> 94
      Figure imgb0046

Claims (18)

  1. A method of identifying a coumarin derivative which exerts an effect onto the activity of a VKORC1 (Vitamine K epoxide reductase complex subunit 1) polypeptide comprising a polypeptide sequence selected from the group consisting of:
    (a) a polypeptide sequence selected from the group consisting of a sequence according to SEQ ID No. 1, 12, and 17;
    (b) a polypeptide sequence of an allele of the polypeptide sequence defined in (a);
    (c) a polypeptide sequence having at least 80% identity with the polypeptide sequence defined in (a) or (b), wherein the polypeptide sequence has VKORC1 activity; and
    (d) a polypeptide sequence of a fragment of the polypeptide sequence defined in (a), (b) or (c) having VKORC1 activity,
    comprising the steps of:
    (I) providing a host cell having been introduced the VKORC1 nucleic acid or a vector containing the VKORC1 nucleic acid;
    (II) expressing the VKORC1 polypeptide in the host cell;
    (III) administering a candidate coumarin derivative;
    (IV) determining the activity of VKORC1 polypeptide (candidate activity value);
    (V) comparing the candidate activity value with a control activity value; and
    (VI) identifying the candidate coumarin derivative as a coumarin derivative exerting an effect onto the activity of the VKORC1 polypeptide, provided the candidate activity value is significantly different from the control activity value.
  2. The method of claim 1, wherein the control activity value is determined by a method comprising the steps of:
    (A) providing a host cell according to step (I);
    (B) expressing the VKORC1 polypeptide in the host cell; and
    (C) determining the activity of VKORC1 polypeptide (control activity value).
  3. The method of claim 1, wherein the determined activity of VKORC1 polypeptide is dithiothreitol-dependent conversion of vitamin K 2,3-epoxide to vitamin K quinone and wherein the significantly different activity value is a candidate activity value which is significantly higher than the control activity value.
  4. The method of claim 1, wherein at least one additional compound is introduced into the host cell, which compound is selected from the group consisting of vitamin K, cytochrome B5, and a nucleic acid coding for gamma-glutamyl-carboxylase, for microsomal epoxidehydrolase, for calumenin, or for glutathion-S-transferase.
  5. A method of determining a VKORC1 polypeptide sequence which conveys a coumarin effect exerted onto VKORC1 activity, comprising the steps of:
    (I) providing a cell expressing a VKORC1 polypeptide comprising a polypeptide sequence selected from the group consisting of:
    (a) a polypeptide sequence selected from the group consisting of a sequence according to SEQ ID No. 1, 12, and 17;
    (b) a polypeptide sequence of an allele of the polypeptide sequence defined in (a);
    (c) a polypeptide sequence having at least 80% identity with the polypeptide sequence defined in (a) or (b), wherein the polypeptide sequence has VKORC1 activity; and
    (d) a polypeptide sequence of a fragment of the polypeptide sequence defined in (a), (b) or (c) having VKORC1 activity, which VKORC1 polypeptide has at least one sequence abnormality;
    (II) administering coumarin or a derivative thereof to the cell;
    (III) determining the activity of the VKORC1 polypeptide (sequence abnormality activity value); and
    (IV) comparing the sequence abnormality activity value with the control sequence activity value,
    wherein a significant deviation of the sequence abnormality activity value from the control sequence activity value is indicative that the sequence abnormality of the VKORC1 polypeptide conveys the coumarin effect exerted onto VKORC1 polypeptide.
  6. The method of claim 5, wherein the control sequence activity value is determined by a method comprising the steps of:
    (I) providing a cell expressing a VKORC1 polypeptide comprising a polypeptide sequence selected from the group consisting of:
    (a) a polypeptide sequence selected from the group consisting of a sequence according to SEQ ID No. 1, 12, and 17;
    (b) a polypeptide sequence of an allele of the polypeptide sequence defined in (a);
    (c) a polypeptide sequence having at least 80% identity with the polypeptide sequence defined in (a) or (b), wherein the polypeptide sequence has VKORC1 activity; and
    (d) a polypeptide sequence of a fragment of the polypeptide sequence defined in (a), (b) or (c) having VKORC1 activity;
    (II) administering coumarin or a derivative thereof to the cell;
    (III) determining the activity of the VKORC1 polypeptide (control sequence activity value).
  7. The method of determining of claim 5, wherein the determined activity is dithiothreitol-dependent conversion of vitamin K 2,3-epoxide to vitamin K quinone and wherein the significantly different value is a sequence abnormality activity value which is significantly higher than the control sequence activity value.
  8. The method of claim 5, wherein at least one additional compound is introduced into the cell which compound is selected from the group consisting of vitamin K, cytochrome B5, and a nucleic acid coding for gamma-glutamyl-carboxylase, for microsomal epoxidehydrolase, for calumenin, or for glutathion-S-transferase.
  9. A VKORC1 polypeptide, wherein the VKORC1 polypeptide contains at least one sequence abnormality, which exerts an effect on the activity of the VKORC1 polypeptide, and wherein the VKORC1 polypeptide is the polypeptide according to SEQ ID No. 1 or 12 and the sequence abnormality is selected from the group consisting of V29L, V45A, R58G, R98W, L128R, and Y139C.
  10. A method of diagnosing warfarin resistance or multiple coagulation factor deficiency type 2 (VKCFD2) in a patient comprising the steps of:
    (I) amplifying a DNA sample obtained from the patient or reverse transcribing a RNA sample obtained from the patient into a DNA and amplifying the DNA; and
    (II) analyzing the amplified DNA of step (I) to determine at least one sequence abnormality in a nucleic acid sequence coding for a VKORC1 polypeptide or in an amino acid sequence of a VKORC1 polypeptide;
    wherein the determined sequence abnormality is indicative of the patient suffering from a VKORC1 associated deficiency; preferably warfarin resistance.
  11. The method of claim 10, wherein the amplified DNA encodes at least a partial sequence of the VKORC1 polypeptide according to SEQ ID No. 1 and wherein the sequence abnormality is selected from the group consisting of V29L, V45A, R58G, R98W, L128R, and Y139C.
  12. The method of claim 10, wherein the amplified DNA is analyzed by a technique selected from the group consisting of PCR- based analysis, restriction digestion analysis, and DNA sequencing analysis.
  13. A method of diagnosing warfarin resistance or multiple coagulation factor deficiency type 2 (VKCFD2) in a patient comprising the steps of:
    (I) providing a sample obtained from the patient; and
    (II) detecting a VKORC1 polypeptide having a sequence abnormality in the sample using antibody or a fragment thereof, which specifically recognizes and binds the VKORC1 polypeptide of claim 9,
    wherein the determined sequence abnormality is indicative of the patient suffering from a VKORC1 associated deficiency.
  14. The method of claim 13, wherein the sample is analyzed by a technique selected from the group consisting of immunohistochemical detection, immunoblotting, preferably Western blotting, and ELISA.
  15. A method of identifying a coumarin derivative which exerts an inhibitory effect onto the activity of a VKORC1 polypeptide having at least one sequence abnormality, comprising the steps of:
    (I) providing a cell expressing the VKORC1 polypeptide according to claim 9, preferably a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No. 3, 4, 5, 6, 7, 14, and 94;
    (II) administering a candidate coumarin derivative to the cell;
    (III) determining the activity of the VKORC1 polypeptide (sequence abnormality activity value); and
    (IV) comparing the sequence abnormality activity value with the control sequence activity value,
    (V) identifying the candidate coumarin derivative as the coumarin derivative exerting an inhibitory effect onto the activity of a VKORC1 polypeptide, if the administration of the candidate coumarin derivative results in a sequence abnormality activity value which is significantly lower than the control sequence activity value.
  16. A method of identifying a coumarin derivative which is toxicologically effective in warfarin-resistant rodents comprising the steps of:
    (I) providing a warfarin-resistant rodent;
    (II) administering a candidate coumarin derivative to the rodent;
    (III) determining the toxicity of the candidate coumarin derivative onto the rodent (candidate coumarin derivative toxicity value);
    (IV) comparing the candidate coumarin derivative toxicity value with a control coumarin toxicity value;
    (V) identifying the candidate coumarin derivative as a toxicologically effective coumarin derivative, provided that the candidate coumarin derivative toxicity value is significantly larger than the control coumarin toxicity value,
    wherein warfarin-resistant rodent is a rodent transgenic for a VKORC1 polypeptide comprising a polypeptide sequence selected from the group consisting of:
    (a) a polypeptide sequence selected from the group consisting of a sequence according to SEQ ID No. 1, 12, and 17;
    (b) a polypeptide sequence of an allele of the polypeptide sequence defined in (a);
    (c) a polypeptide sequence having at least 80% identity with the polypeptide sequence defined in (a) or (b), wherein the polypeptide sequence has VKORC1 activity; and
    (d) a polypeptide sequence of a fragment of the polypeptide sequence defined in (a), (b) or (c) having VKORC1 activity, wherein the VKORC1 polypeptide contains at least one sequence abnormality, which causes warfarin resistance, preferably a polypeptide encoded by the sequence according to SEQ ID No. 14.
  17. The method of claim 16, wherein VKORC1 polypeptide is the polypeptide according to SEQ ID No. 12 and the sequence abnormality is selected from the group consisting of V29L (85 G>T), V45A(134 T>C), R58G (172 A>G), R98W (292 C>T), and L128R (383 T>G), and Y139C (416 A>G).).
  18. Use of the PCR primers according to SEQ ID No. 88 to 91 for determining whether or not a rat has a warfarin resistance genotype in a sample obtained from a rat.
EP04790318A 2003-10-14 2004-10-12 Vitamin k epoxide recycling polypeptide vkorc1, a therapeutic target of coumarin and their derivatives Not-in-force EP1673450B1 (en)

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PL04790318T PL1673450T3 (en) 2003-10-14 2004-10-12 Vitamin k epoxide recycling polypeptide vkorc1, a therapeutic target of coumarin and their derivatives
EP10002316A EP2189523B1 (en) 2003-10-14 2004-10-12 Vitamin K epoxide recycling polypeptide VKORC1, a therapeutic target of coumarin and their derivatives
DK10002316.7T DK2189523T3 (en) 2003-10-14 2004-10-12 VKORC1 (vitamin K epoxide feedback polypeptide), a therapeutic target for coumarin and its derivatives
PL10002316T PL2189523T3 (en) 2003-10-14 2004-10-12 Vitamin K epoxide recycling polypeptide VKORC1, a therapeutic target of coumarin and their derivatives
EP10011278.8A EP2272951B1 (en) 2003-10-14 2004-10-12 Vitamin k epoxide recycling polypeptide vkorc1, a therapeutic target of coumarin and their derivatives

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